BIOLOGY 

LIBRARY 

G 


THE  METABOLISM  OF  BILE  ACIDS 


A  THESIS  ACCEPTED  IN  PARTIAL  SATISFACTION  OF 
THE  REQUIREMENTS  FOR  THE  DEGREE  OF 

DOCTOR  OF  PHILOSOPHY 
AT  THE  UNIVERSITY  OF  CALIFORNIA 


BY 


MARJORIE  GREENE  FOSTER. 


1919 


THE  METABOLISM  OF  BILE  ACIDS 

I.    A  QUANTITATIVE  METHOD   FOR  ANALYSIS  OF 
BILE  ACIDS  IN  DOG'S  BILE 


BY 

M.  G.  FOSTER  AND  C.  W.  HOOPER 


(FROM  THE  GEORGE  WILLIAMS  HOOPER  FOUNDATION  FOR  MEDICAL 

RESEARCH,  UNIVERSITY  OF  CALIFORNIA  MEDICAL  SCHOOL, 

SAN  FRANCISCO) 


REPRINTED  FROM 

THE  JOURNAL  OF  BIOLOGICAL  CHEMISTRY 
VOL.  XXXVIII,  No.  2,  JUNE,  1919 


B/OLOGY 

UBRfi  RY 

G 


Reprinted  from  THE  JOURNAL  OF  BIOLOGICAL  CHEMISTRY,  Vol.  XXSVil1.,  No.  2,  lc»l&. 


THE  METABOLISM  OF  BILE  ACIDS. 

I.     A  QUANTITATIVE  METHOD  FOR  ANALYSIS  OF  BILE  ACIDS 
IN  DOG'S  BILE. 

BY  M.  G.  FOSTER*  AND  C.  W.  HOOPER. 

(From  the  George  Williams  Hooper  Foundation  for  Medical  Research,  Uni- 
versity of  California  Medical  School,  San  Francisco.} 

(Received  for,  publication,  April  28,  1919.) 

An  unusual  opportunity  for  a  comprehensive  study  of  the  bile 
acids  presented  itself  to  us  in  this  laboratory  because  of  the  pres- 
ence of  a  number  of  bile  fistula  dogs  under  careful  routine 
observation.  These  bile  fistula  dogs  were  under  observation 
for  the  study  of  bile  pigments.  They  present  normal  factors 
of  weight,  activity,  and  appetite  when  under  the  laboratory 
routine  care  which  has  been  described  in  detail  by  Hooper  and 
Whipple  (8). 

In  order  to  study  satisfactorily  the  metabolism  of  the  bile 
acids  it  is  necessary  to  have  a  method  for  the  analysis  of  bile 
acids  which  is  relatively  simple  and  accurate  and  which  does  not 
require  large  quantities  of  material  for  analysis.  The  method 
must  be  specific  for  bile  acids  and  react  negatively  with  the  other 
substances  in  bile  and  it  must  not  be  obscured  by  any  substances 
which  may  be  present  in  bile  fistula  bile.  We  know  of  no  pub- 
lished method  which  meets  all  these  requirements. 

As  a  preliminary  step  we  undertook  to  test  a  number  of  the 
clinical  methods  for  the  determination  of  bile  acids,  some  of  which 
are  known  to  be  inaccurate  and  others  of  which  are  claimed  to 
be  merely  qualitative.  Without  exception  we  have  found  these 

*This  series  of  papers  on  Bile  Acid  Metabolism  was  completed  just 
prior  to  the  death  of  Miss  Foster  from  influenza  pneumonia.  The  work 
should  stand  as  a  memorial  to  her  enthusiasm,  patience,  and  spirit  of  truth- 
ful research.  This  work  was  submitted  as  a  thesis  for  her  degree  of  Doctor 
of  Philosophy,  University  of  California. 

355 


356" 


»        «   *    • 

Metabolism  of  Bile  Acids.    I 


•V.:  /:>:•'•";:    :  A  '': 

feMrvdy  'siiiipfe  elmical'  methods  to  be  grossly  inaccurate  and 
useless  for  analysis  of  bile  salts.  This  statement  applies  to  a  re- 
cent method  advocated  by  Hoover  and  Blankenhorn  (9).  No 
controls  are  given  by  these  workers  to  show  that  many  other 
substances  present  in  abnormal  sera  may  not  have  been  respon- 
sible for  the  positive  bile  acid  reaction  noted  in  their  experiments. 
For  a  discussion  of  these  possibilities  refer  to  the  paragraph  below 
on  Pettenkofer's  test. 

Some  of  the  more  elaborate  chemical  methods  for  the  ex- 
traction of  bile  salts  from  bile  are'  relatively  accurate,  but  the 
large  quantity  of  material  used  and  the  amount  of  time  required 
make  frequent  analyses  at  short  intervals  impracticable.  These 
extraction  methods  are  very  expensive  as,  well  as  time-consuming. 
The  older  methods  may  be  divided  into  three  groups  :  (1)  methods 
in  which  the  bile  acids  are  separated  and  weighed;  (2)  methods 
in  which  the  bile  acids  are  calculated  from  the  sulfur  content 
in  whole  bile;  and  (3)  methods  which  depend  on  the  color  re- 
actions of  cholic  acid.*  There  are  possibilities  of  error  in  all  these 
methods,  as  will  be  pointed  out  later. 

Methods  in  Which  the  Bile  Acids  are  Separated  and  Weighed. 

Huppert's  Method  —  1864.  —  Huppert  (11)  modified  Neukomm's  (17)  meth- 
od for  bile  salts  in  urine  and  applied  it  to  the  bile  and  blood  in  the 
following  way.  The  albumin  was  first  coagulated  with  alcohol  and  the 
precipitate  carefully  extracted  with  additional  amounts  of  alcohol.  The 
whole  extract  was  evaporated,  dissolved  in  water,  freed  from  fat  by  ether 
extraction,  neutralized,  and  Ba(N03)2  added  to  separate  the  fatty  acids, 
soaps,  and  any  remaining  protein.  The  washed  precipitate  was  supposedly 
free  from  bile  acids.  The  filtrate  and  wash  water  were  precipitated 
with  lead  acetate  and  the  precipitate  washed  with  water  to  free  from  excess 
acetate.  The  precipitate  was  then  washed  out  with  alcohol,  heated,  and 
NagCOa  added.  After  it  was  evaporated  to  dryness,  the  residue  was  ex- 
tracted with  absolute  alcohol;  the  alcohol  was  evaporated  to  dryness;  and 
the  residue  was  dissolved  in  water.  This  water  solution  was  filtered  into 
a  weighed  dish  and  dried  to  constant  weight. 

Socoloff's  Method—  1875.  —  Socoloff  (19)  published  a  method  in  which  the 
bile  acids  present  were  calculated  by  the  amount  of  bile  soluble  in  alcohol. 
This  work  was  done  in  Hoppe-Seyler's  laboratory  using  a  method  very 
much  like  the  one  described  in  the  following  paragraph. 

Hoppe-Seyler's  Method  —  1881.  —  Hoppe-Seyler's  method  (10)  was  used 
by  Pfaff  and  Balch,  Stadelmann,  and  others.  The  bile  was  dried  to  con- 
stant weight  at  110°C.  and  the  residue  completely  extracted  with  boiling 


M.  G.  Foster  and  C.  W.  Hooper  357 

absolute  alcohol.  The  extract  was  allowed  to  stand  24  hours,  filtered,  and 
evaporated  to  dryness  at  110°C.  The  residue  was  dissolved  in  absolute 
alcohol,  evaporated  to  a  small  volume,  cooled,  and  precipitated  with  a 
large  excess  of  absolute  ether.  The  bile  salts  which  crystallized  out  were 
dried  at  110°C.  and  weighed.  Stadelmann  (21)  evaporated  the  bile  to  a 
thick  syrup  and  allowed  it  to  stand  in  contact  with  alcohol  on  the  water 
bath  some  time  in  order  to  coagulate  the  protein  and  give  a  clear  filtrate. 
He  then  repeated  the  extraction  three  times  with  boiling  96  per  cent  alcohol. 
He  dissolved  the  final  precipitate  in  water  and  dried  to  constant  weight. 

Croftan's  Method— 1902—  Croftan  (3,  4)  estimated  the  bile  acids  by. 
coagulating  the  albumin,  evaporating  the  filtrate  and  washings,  and  pre- 
cipitating with  absolute  alcohol  to  free  from  salts.  The  filtrate  was  diluted 
with  water,  precipitated  with  basic  lead  acetate  and  ammonia,  and  the 
precipitate  extracted  with  absolute  alcohol  and  filtered  hot.  This  solution 
of  bile  salts  was  dried  to  constant  weight.  (According  to  Croftan  the  mucin 
holds  with  it  a  large  amount  of  bile  acid  and  they  are  so  closely  bound  that 
it  is  impossible  to  separate  them  even  by  repeated  washings.) 

Goodman's  Method — 1906. — Goodman  (5)  hydrolyzed  50  gm.  of  bile  with 
125  gm.of  60  per  cent  KOH  in  a  reflux  condenser  for  24  hours.  The  solu- 
tion was  extracted  five  times  with  75  cc.  of  freshly  distilled  petroleum  ether 
in  order  to  extract  the  cholesterol.  The  last  of  the  petroleum  ether  was 
evaporated  off  on  a  water  bath  and  the  solution  precipitated  with  5  per 
cent  BaCl2  to  separate  the  higher  fatty  acids.  The  precipitate  was  ex- 
tracted with  boiling  water  and  the  filtrate  and  washings  evaporated  to 
200  to  300  cc.  The  solution  was  cooled  in  ice  and  salt,  and  acidified  with 
HC1.  After  standing  2  hours  the  cholic  acid  which  crystallized  out  was 
filtered  off,  washed,  dried,  and  extracted  in  a  Soxhlet  apparatus  with  ace- 
tone. In  about  5  hours  all  the  cholic  acid  was  dissolved  and  the  solution 
was  evaporated  and  dried  to  constant  weight. 

Methods  in  Which  the  Bile  Acids  Are  Calculated  from  the  Sulfur  Content  in 

Whole  Bile. 

Spiro's  Method— 1880.— The  sulfur  method  as  used  by  Spiro  (20)  con- 
sisted in  fusing  a  definite  amount  of  bile  (usually  50  cc.)  in  a  silver  dish 
with  KOH  and  KNO3.  The  mixture  was  dissolved  in  water,  supersatu- 
rated with  HC1,  and  precipitated  hot  with  BaCl2.  This  precipitate  was 
filtered  on  a  paper  of  known  ash,  washed  well,  and  dried.  The  main  part 
of  the  precipitate  was  transferred  to  a  porcelain  crucible.  The  paper 
with  the  remainder  of  the  precipitate  was  ignited  and  weighed.  The  main 
part  of  the  precipitate  was  weighed,  a  few  drops  of  concentrated  H2SO4 
added,  washed  out  well  with  water  into  another  filter  paper,  and  this  was 
weighed.  The  difference  in  weight  showed  the  amount  of  the  impurity. 
From  this  difference  was  calculated  the  BaSO4  in  the  other  portion  of  the 
precipitate  which  was  weighed  with  the  ash  of  the  larger  filter  paper,  and 
the  total  amount  of  sulfur  was  calculated  from  this  corrected  value. 


358  Metabolism  of  Bile  Acids.     I 

Von  Bergman's  Method — 1904- — Von  Bergman  (2)  precipitated  a  24  hour 
collection  of  bile  with  many  volumes  of  96  per  cent  alcohol.  The  mu- 
cin  precipitate  was  filtered  off  and  washed  six  or  eight  times  with  alcohol. 
The  combined  washings  and  filtrate  were  evaporated  to  a  definite  volume 
and  an  aliquot  part  used  for  the  determination  of  the  combined  sulfur. 

Methods  Based  on  the  Color  Reactions  of  Cholic  Acid. 

Pettenkofer's  Method. — This  test  as  outlined  in  Hammarsten  (6)  is  per- 
formed by  dissolving  a  small  amount  of  bile  in  concentrated  sulfuric  acid 
and  warming  to  60  or  70°C.  A  10  per  cent  solution  of  cane  sugar  is  added 
drop  by  drop.  A  beautiful  red  color  develops  which  turns  bluish  violet 
on  standing.  The  red  liquid  shows  a  spectrum  with  two  absorption  bands, 
one  at  F  and  one  between  D  and  E  .  The  test  fails  if  the  solution  is  heated 
too  hot,  or  if  too  much  sugar  is  added  (on  account  of  the  sugar  carboniz- 
ing). Also,  if  impurities  are  present  in  the  acid,  such  as  H2SO3  or  the 
lower  oxides  of  nitrogen,  the  reaction  fails.  Proteins,  amyl  alcohol,  oleic 
acid,  morphine,  etc.,  give  a  similar  color  so  that  it  is  necessary  to  carry  out 
the  spectroscopic  examination  also.  Ville  and  Deriien  (23)  state  that 
vanillin  and  anisaldehyde  give  the  same  color,  and  cholesterol  gives  a  sim- 
ilar color.  This  method  has  been  improved  upon  by  Mylius  and  von  Udran- 
szky,  who  advise  the  use  of  a  1  per  cent  solution  of  furfurol  in  place  of  the 
sugar.  Von  Udranszky  (22)  emphasizes  the  fact  that  pure  bile  acids  are 
necessary  and  suggests  decolorizing  the  bile  with  charcoal  and  using  an 
alcoholic  solution  of  the  residue.  To  each  cc.  of  alcoholic  solution,  add  one 
drop  of  furfurol  and  1  cc.  of  concentrated  sulfuric  acid,  and  warm  gently, 
This  will  detect  0*0  to  ^V  mg.  of  cholic  acid.  According  to  Hammarsten,  the 
protein  and  fat  should  also  be  removed  by  neutralizing  the  bile  and  adding 
alcohol  to  at  least  85  volumes  per  cent  pure  alcohol.  The  solution  is  fil- 
tered and  the  protein  extracted-  with  fresh  alcohol,  and  the  alcoholic  ex- 
tract evaporated  to  dryness.  This  residue  is  extracted  with  absolute 
alcohol,  filtered,  and  the  extract  evaporated  to  dryness.  The  residue  is 
extracted  with  ether,  dissolved  in  water,  and  the  solution  precipitated  with 
basic  lead  acetate  and  NH4OH.  The  precipitate  is  washed  and  dissolved 
in  boiling  alcohol,  filtered,  and  made  alkaline  with  a  few  drops  of  ^SaOH. 
This  solution  is  evaporated  to  dryness,  the  residue  extracted  with  absolute 
alcohol,  filtered,  and  precipitated  with  ether.  This  solution  may  be  used 
for  Pettenkofer's  test,  but  even  this  may  contain  phosphatides  and  they 
give  the  same  color  reaction  as  do  the  bile  acids. 

Inouye  and  Ito  (12)  reported  that  when  vanillin  and  concentrated  sul- 
furic acid  are  added  to  solutions  of  bile  acids  a  red  line  is  formed  at  the 
line  of  contact.  If  the  fluids  are  then  mixed,  the  solution  changes  to  a 
red-brown  and  then  violet.  When  this  solution  is  diluted  with  glacial  ace- 
tic acid,  an  adsorption  band  is  shown  at  B.  This  reaction  is  sensitive 
with  taurocholic  acid  in  a  dilution  of  1 : 11,000  and  with  cholic  acid  1 : 22,000. 
Jolles  (13)  published  a  method  in  which  the  bile  acid  (2  to  3  cc.  of  1  per 
cent  solution)  is  mixed  with  rhamnose  (1  to  2  drops  of  5  per  cent  solution) 


M.  G.  Foster  and  C.  W.  Hooper  359 

and  boiled  with*2  to  3  cc.  of  concentrated  HC1.  A  red  color  is  produced  fol- 
lowed by  a  green  fluorescence  which  is  due  to  the  formation  of  methyl  fur- 
furol  aldehyde.  The  reaction  can  be  carried  out  by  0.005  to  0.0001  gm. 
of  pure  acid.  It  is  not  affected  by  urea,  albumin,  carbohydrates,  hydro- 
carbons, or  acids  of  the  aliphatic  or  aromatic  series,  glycocoll,  taurine, 
or  cholesterol. 

Authors'  Method. 

Dog's  bile  contains  only  taurocholic  and  taurocholeic  acids, 
and  on  hydrolysis  these  split  into  taurine  and  cholic  and  choleic 
acids.  Taurine  is  amino  ethyl  sulfonic  acid,  CH2NH2CH2HSO3. 
It  acts  like  an  a-amino-acid,  and  gives  off  its  NH2  quantitatively 
in  3  minutes  in  the  Van  Slyke  amino  nitrogen  apparatus. 
This  method  consists  in  hydrolyzing  a  definite  aniount  of  bile 
with  NaOH,  thus  splitting  the  bile  acid,  and  then  determining 
the  amount  of  NH2  in  the  taurine.  Neither  taurocholic  nor 
taurocholeic  acid  gives  off  NH2  before  hydrolysis.  In  other 
animals  both  taurocholic  and  glycocholic  acids  are  present,  and 
it  is  therefore  not  certain  that  the  method  in  its  present  form  can 
be  applied  to  the  bile  of  animals  other  than  the  dog. 

For  the  determination  5  cc.  of  bile  cleared  in  the  centrifuge 
and  measured  in  a  calibrated  pipette  are  precipitated  with  40  cc. 
of  95  per  cent  alcohol,  and  heated  to  the  boiling  point  to  insure 
a  complete  solution  of  the  bile  acid  which  might  be  held  with 
the  mucin  precipitate.  After  cooling,  the  mixture  is  made  up 
to  50  cc.  in  a  cylinder  with  95  per  cent  alcohol,  and  passed  through 
a  dry  filter  paper.  Two  specimens  of  20  cc.  each  are  evaporated 
to  dryness.  One  is  washed  out  with  water  and  made  up  to  10  cc. 
in  a  calibrated  flask.  2  cc.  samples  of  this  are  used  to  determine 
the  amount  of  NH2  present  before  hydrolysis.  The  other  is 
washed  out  quantitatively  with  6  cc.  of  8  per  cent  NaOH  into 
a  test-tube.  The  test-tube  is  loosely  stoppered  and  placed  in 
a  boiling  water  bath  for  5  hours.  The  contents  are  washed  out 
into  a  10  cc.  calibrated  flask  and  made  up  to  volume  with  distilled 
water.  2  cc.  samples  of  this  are  used  to  determine  the  amount 
of  amino  nitrogen  due  to  the  hydrolysis  of  the  bile  acids.  The 
nitrogen  is  then  figured  on  the  basis  of  1  cc.  of  bile  for  both  speci- 
mens by  multiplying  the  mg.  of  amino  nitrogen  found  by  2.5 
and  subtracting  the  unhydrolyzed  amino  nitrogen  from  that  due 
to  hydrolysis.  This  figure  is  multiplied  by  the  total  volume  of 


360 


Metabolism  of  Bile  Acids.     I 


bile  for  the  6  hour  collection,  which  gives  the  total  output  of 
amino  nitrogen  for  6  hours.  The  bile  acids  are  figured  as  tauro- 
cholic  acid  by  multiplying  by  36.72,  the  factor  obtained  by  divid- 
ing the  molecular  weight  of  taurocholic  acid  by  the  atomic  weight 
of  nitrogen. 

Quantitative  Estimation  of  Taurine. 

Table  I  shows  that  taurine  gives  up  its  NH2  quantitatively 
with  3  minutes  shaking  in  the  Van  Slyke  amino  nitrogen  appara- 
tus. The  reaction  is  complete  in  that  time  even  at  as  low  a  tem- 
perature as  13°C. 

TABLE  I. 

Quantitative  Estimation  of  Taurine. 


Taurine. 

Nitrogen 
gas. 

Temper- 
ature. 

Pressure. 

Correction. 

NHz-N 
found. 

NH2-N 
theoretical. 

mg. 

cc. 

°C. 

mm. 

cc. 

mg. 

mg. 

46.00 

9.11 

18 

760 

0.10 

5.157 

5.152 

9.10 

9.785 

2.06 

22 

.    756 

0.10 

1.095 

1.096 

2.07 

10.58 

2.17 

13 

759 

0.14 

1.189 

1.185 

2.17 

Analysis  of  Sodium  Taurocholate. 

To  test  out  the  method  a  specimen  of  sodium  taurocholate  was 
analyzed  for  N,  S,  and  ash,  and  the  NH2  yielded  after  hydroly- 
sis. Table  II  shows  that  the  content  of  both  N  and  S  of  the 
sodium  taurocholate  used  was  about  85  per  cent  of  their  theoreti- 
cal values.  The  13.3  per  cent  ash  partly  accounts  for  the  low 
values  of  N  and  S.  Sodium  taurocholate  should  have  had  only 
4.3  per  cent  sodium  in  the  ash  if  one  hydrogen  atom  was  replaced 
by  Na. 

The  solutions  used  in  Table  III  were  hydrolyzed  5  hours  in 
a  boiling  water  bath  and  made  up  to  10  cc.  in  a  calibrated  flask. 
NH2  determinations  gave  the  following  results. 


M.  G.  Foster  and  C.  W.  Hooper 


361 


Number. 

Amount 
used. 

Nitrogen 
gas. 

Temper- 
ature. 

Pressure. 

Correc- 
tion. 

Amino 
nitrogen 
in  10  cc. 
solution. 

Total 
NH?-N 
in  10  cc. 

Hydro- 
lysis 
NH2 

"11" 

cc. 

cc. 

°C. 

mm. 

cc. 

mg. 

mg. 

per  cent 

1 

I 

1.83 

20 

758 

0.10 

9.74 

10.29 

94.7 

1.82 

2 

1 

1.28 

20 

758 

0.10 

6.62 

6.86 

96.5 

1.26 

3 

2 

1.31 

20 

758 

0.10 

3.42 

3.43 

99.8 

1.31 

4 

2 

0.33 

20 

'758 

0.10 

0.65 

0.68 

95.6 

0.33 

TABLE  II. 
Analysis  of  Sodium  Taurocholate. 


Sodium 
taurocholate. 

Total  N. 

Theoretical  N. 

Per  cent  of  theo- 
retical value. 

Ash. 

mg. 

mg. 

per  cent 

per-  cent 

per  cent 

per  cent 

779.6 

17.15 

2.20 

2.60 

84.62 

13.3 

Sodium 
taurocholate. 

BaSO4 

Total  S. 

.  Theoretical  S. 

Per  cent  of 
theoretical  value. 

mg. 

250* 

mg. 

92.6 

per  cent 

5.09 

per  cent 

5.96 

per  cent 

85.42 

*  The  sulfur  determinations  were  very  kindly  carried  out  by  Dr.  C.  L. 
A,  Schmidt. 

TABLE  III. 

Hydrolysis  of  Sodium  Taurocholate. 


Number. 

Sodium  taurocholate 
solution.* 

Water  added. 

Sodium 
taurocholate. 

16  per  rent 
of  NaOH 
added. 

NaOH  in 
solution. 

cc. 

mg. 

cc. 

per  cent 

cc. 

per  cent 

1 

3 

467.7 

0 

15 

3 

8 

2 

2 

311.8 

1 

10 

3 

8 

.  3 

1 

155.9 

2 

6 

3 

8 

4 

3t 

31.2 

•0 

1 

3 

8 

*  3. 8980  gm.  were  made  up  to  volume  in  a  25  cc.  flask  and  definite 
amounts  were  used. 

t  Solution  prepared  by  diluting  1  cc.  of  the  original  solution  with  14 
cc.  of  distilled  water. 


THE   JOURNAL   OF   BIOLOGICAL   CHEMISTRY,    VOL.   XXXVIII,    NO. 


362  Metabolism  of  Bile  Acids.    I 

Table  III  shows  that  the  hydrolysis  of  this  specimen  of  sodium 
taurocholate  in  from  1  to  15  per  cent  solutions  was  approxi- 
mately 100  per  cent  in  5  hours  when  the  alkali  present  was  8  per 
cent  during  the  hydrolysis. 

This  complete  analysis  was  carried  out  on  another  specimen  of 
sodium  taurocholate  with  the  same  results. 

Normal  Constituents  of  Whole  Bile. 

According  to  Hoppe-Seyler  (10)  the  normal  constituents  of 
whole  bile  are  bile  salts,  bile  pigments,  cholesterol,  mucin,  ethereal 
sulfates,  conjugated  glucuronic  acids,  fats,  soaps,  a  trace  of  urea, 
jercorin  and  other  phosphatides,  hydrochloric  and  phosphoric 
acids,  and  sulfuric  acid  as  Na,  P,  Ca,  Mg,  Fe,  and  Cu  salts.  Mar- 
shall and  Davis  (16)  found  the  same  amount  of  urea  in  the  bile 
as  in  the  blood — 32  mg.  of  urea  per  100  cc.  1  cc.  of  bile  would 
then  contain  0.149  mg.  of  N,  but  Van  Slyke  states  that  only  3  per 
cent  of  N  in  the  urea  is  given  off  in  the  first  3  minutes,  i.e.  0.00447 
mg.  of  N,  and  that  amount  is  not  sufficient  to  cause  an  appreciable 
error  in  this  method.  Traces  of  amino-acids  have  been  found 
in  disease,  but  these  would  give  off  their  NH2  nitrogen  in  the 
unhydrolyzed  specimen,  and  thus  would  be  corrected  for.  Stadel- 
mann  thinks  that  glycocholic  acid  may  be  present  in  dog's  bile. 
The  generally  accepted  opinion  is  that  dog's  bile  contains  no 
glycocholic  acid  or  at  the  most  a  small  trace.  Even  the  pres- 
ence of  glycocholic  acid  sufficient  to  make  up  10  per  cent  of  the 
hypothetical  bile  acid  mixture,  would  introduce  no  appreciable 
error,  figuring  all  the  bile  acids  as  taurocholic  acid.  This  is 
because  of  the  large  size  of  the  cholic  acid  molecule  and  the  simi- 
larity of  weight  of  the  molecules  of  glycocol  and  taurine.  None 
of  the  other  constituents  set  free  NH2  groups  on  hydrolysis 
except  mucin.  To  obviate  this  error  we  precipitated  the  mucin 
with  10  volumes  of  95  per  cent  alcohol.  In  one  set  it  was  filtered 
immediately;  in  a  second,  the  alcohol  was  allowed  to  remain  in 
contact  with  the  mucin  for  24  hours;  and  in  a  third,  the  alcoholic 
mixture  was  heated  to  its  boiling  point,  cooled,  and  filtered  im- 
mediately. Since  Croftan  showed  that  the  mucin  precipitate 
could  not  be  freed  of  bile  acid  by  extraction,  we  made  the  mixture 
.to  a  definite  volume  with  alcohol  and  then  took  an  aliquot  part 
of  the  filtrate.  This  would  insure  a  uniform  mixture  of  bile  salts. 


M.  G.  Foster  and  C.  W.  Hooper 


363 


Table  IV  shows  that  muciri  is  best  removed  by  alcohol  heated 
to  its  boiling  point.  The  NH2  given  off  by  the  unhydrolyzed 
bile  may  be  due  to  traces  of  urea  or  to  some  splitting  of  the  bile 
acid.  It  is  always  proportional  to  the  bile  acid  content,  but  not 
in  exact  ratio.  The  unhydrolyzed  mucin-free  bile  gives  a  higher 
NH2  in  some  cases  than  whole  bile.  This  may  be  due  to  a  slight 
splitting  of  the  bile  acid.  The  hydrolyzed  whole  bile  gives  off 
decidedly  more  NH2  nitrogen  than  the  mucin-free  bile.  This 
shows  that  the  mucin  is  decomposed  by  hydrolysis  into  NH2- 

TABLE  IV. 
Whole  Bile  and  Mucin-Free  Bile. 


Dog. 

Unhydrolyzed  bile. 

Hydrolyzed  bile. 

Whole. 

Mucin-free. 

Whole. 

Mucin-free. 

NH2-N*per 
cc.  of  bile. 

NH2-Nper 
cc.  of  bile. 

NH2-Nper 
cc.  of  bile. 

NH2-Nper 
cc.  of  bile 
filtered  im- 
mediately. 

NH2-N  per 

cc.  of  bile  af- 
ter 24  hours. 

NH2^Nper 
cc.  of  bile 
heated  to 
boiling. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg 

17-151 

0.194 

0.245 

1.210 

0.924 

1.012 

0.915 

16-  15 

0.051 

0.072 

0.277 

0.188 

0.144 

0.203 

15-  22 

0.205 

0.245 

1.386 

1.185 

1.214 

1.276 

18-  23 

0.080 

0.072 

0.452 

0.216 

0.289 

0.290 

*  The  readings  on  the  NH2  apparatus  have  been  omitted  for  simplicity. 

containing  substances  (amino-acids) .  The  mucin  precipitated 
by  heating  the  mixture  of  bile  and  alcohol  to  boiling  seems  to 
give  the  quickest  and  most  satisfactory  results. 

Sodium  Taurocholate   Added  to  Bile. 

To  ascertain  if  the  amino  nitrogen  determined  in  hydrolyzed 
bile  is  really  specific  for  the  bile  acid,  three  specimens  of  the  same 
bile  were  hydrolyzed  after  adding  different  known  amounts  of 
sodium  taurocholate  to  each.  A  control  was  run  on  the  bile 
and  on  the  sodium  taurocholate  solution. 

Table  V  shows  that  known  amounts  of  sodium  taurocholate 
added  to  bile  can  be  recovered  quantitatively  with  this  method. 
The  theoretical  increase  is  figured  from  No.  1.  The  error  is 
about  6  to  8  per  cent  loss.  Since  our  conclusions  are  based  on 


364 


Metabolism  of  Bile  Acids.     I 


decided  increases  in  bile  acid  excretion  in  our  experiments,  a 
loss  due  to  the  method  would  have  little  significance  in  the  analy- 
sis of  results. 

TABLE  V. 
Known  Amounts  of  Sodium  Taurocholate  Added  to  Bile. 


Number. 

Bile. 

Sodium 
taurocholate. 
(15  per  cent 
solution). 

NH2-N 

increase. 

NH2-N  in- 
crease due 
to  sodium 
taurocholate. 

Theoretical 
increase. 

Error  (loss). 

cc. 

cc. 

mg.* 

mg. 

mg. 

p*r  cent 

1 

0 

1 

1.150 

2 

5 

3 

4.061 

3.229 

3.45 

6.4 

3 

5 

2 

2.953 

2.121 

2.30 

7.8 

4 

5 

1 

1.914 

1.082 

1.15 

6.1 

5 

5 

0 

0.832 

*  The  readings  for  the  NH2  have  been  omitted  for  simplicity. 

A  Six  Hour  Collection  of  Bile. 

Table  VI  shows  the  same  total  for  three  periods  of  2  hours 
each  as  for  one  period  of  6  hours.  This  speaks  for  a  thorough 
mixture  of  the  thick  viscous  bile  and  its  contained  bile  acids  and 
shows  that  a  single  estimation  is  accurate  for  the  total  excretion. 
It  also  gives  confirmatory  evidence  of  the  accuracy  of  this  method. 

TABLE  VI. 
Mixed  Collection  of  Bile. 


Time. 

NH2-N  per  1 

Volume. 

Total  NHz. 

Output. 

Per  6  hours. 

hrs. 

mg. 

cc. 

mg. 

mg. 

1-2 

1.174 

15 

17.61 

3^ 

0.553 

14 

7.74 

£-6 

0.558 

16 

8.93 

34.  28  Total. 

1-6 

0.753 

45 

33.88 

33.  88  Total. 

Variations  in  the  Concentration  of  Alkali. 

The  amount  and  concentration  of  alkali  used  are  of  minor  im- 
portance as  can  be  seen  in  Table  VII.  The  same  specimen  of 
bile  was  hydrolyzed  with  varying  amounts  and  concentration  of 


M.  G.  Foster  and  C.  W.  Hooper 


365 


NaOH  and  the  results  show 'that  the  bile  acid  was  completely 
hydrolyzed  in  all  the  tubes.  Table  VII  also  shows  that  slight 
variations  in  the  amount  and  concentration  of  alkali  used  for 
the  hydrolysis  do  not  affect  the  accuracy  of  the  method. 

TABLE  VII. 
Strength  of  NaOH  Used. 


Tube. 

NaOH 

NH2-N 

cc. 

per  cent 

mg. 

1 

5 

8 

0.364 

2 

6 

8 

0.364 

3 

7 

8 

0.350 

4 

6 

6 

0.350 

5 

6 

12 

0.364 

DISCUSSION. 

Hammarsten  (6  and  7)  found  jecorin,  lecithin,  and  other  phos- 
phatides  in  bile,  and  all  contain  nitrogen  and  sulfur.  Since  the 
nitrogen  is  in  the  choline  radical,  it  does  not  interfere  with  the 
present  method,  as  choline  on  hydrolysis  with  alkalies  yields 
trimethylamine  and  glycol,  and  trimethylamine  does  not  react 
with  nitrous  acid.  But  sulfur  does  interfere  with  the  bile  acid 
methods  as  determined  by  the  sulfur  content. 

The  phospha tides  cause  a  decided  error  in  the  methods  in  which 
the  bile  acids  are  weighed.  Long  and  Gephart  (15)  have  found 
it .  impossible  to  separate  bile  acids  from  lecithin  even  with  ace- 
tone. Bile  salts  can  hold  in  stable  solution  80  per  cent  of  their 
weight  of  egg  lecithin.  Part  of  this  can  be  separated  by  pre- 
cipitation, but  the  amount  remaining  with  the  bile  acids  and  not 
separated  by  acetone  is  much  in  excess  of  that  contained  in  any 
bile.  Ethereal  sulfates  are  present  in  some  bile  (human  and 
shark)  according  to  Hammarsten,  and  thus  interfere  with  the 
sulfur  determination.  But  von  Bergman  could  not  detect  any 
in  dog's  bile.  It  is  evident  that  there  are  many  sources  of  error 
in  all  the  older  methods. 

The  method  outlined  in  this  paper  is  not  open  to  the  criticisms 
of  the  methods  previously  used  for  the  quantitative  estimation 
of  the  bile  salts.  It  is  a  simple  procedure  requiring  careful  tech- 
nique only  in  washing  out  the  various  residues  and  making  up  to 
volume. 


366  Metabolism  of  Bile  Acids.     I 

The  results,  although  6  to  8  per  cent  below  the  theoretical 
values,  are  very  constant,  as  we  have  demonstrated  over  and  over 
again  when  duplicates  were  run  through  by  several  different 
people.  The  determinations  can  be  carried  out  so  that  the  results 
are  available  within  8  hours. 

SUMMARY. 

A  method  is  given  for  the  quantitative  estimation  of  the  bile 
acid  present  in  dog's  bile.  It  is  based  on  the  fact  that  taurine 
gives  up  its  NH2  nitrogen  quantitatively  in  the  Van  Slyke  ammo 
nitrogen  apparatus.  The  taurocholic  acid  is  hydrolyzed  by  NaOH 
into  taurine  and  cholic  acid.  The  amino  nitrogen  of  the  taurine 
is  then  determined  by  the  gasometric  method. 

We  are  indebted  to  both  Dr.  Alice  Rohde  and  Dr.  Donald 
D.  Van  Slyke  for  their  advice  and  help  in  working  out  this  method; 
also  to  Dr.  C.  L.  A.  Schmidt  for  sulfur  determinations. 

BIBLIOGRAPHY. 

1.  Beddard,  A.  P.,  and  Pemhrey,  M.  S.,  Brit  Med.  J.,  1902,  i,  702. 

2.  von  Bergman,  G.,  Beit.  chem.  Phys.  u.  Path.,  1904,  iv,  192. 

3.  Croftan,  A.  C.,  Am.  J.  Med.  Sc.,  1902,  cxxiii,  150. 

4.  Croftan,  A.  C.,  Phil.  Med.  J.,  1902,  ix,  75,  142. 

5.  Goodman,  E.  H.,  Beitr.-chem.  Phys.  u.  Path.,  1907,  ix,  91. 

6.  Hammarsten,  O.,  text  book  of  physiological  chemistry,  New  York,  7th 

edition,  1914. 

7.  Hammarsten,  O.,  Ergebn.  Physiol.,  1905,  iv,  7. 

8.  Hooper,  C.  W.,  and  Whipple,  G.  H.,  Am.  J.  Physiol.,  1916,  xl,  332. 

9.  Hoover,  C.  F.,  and  Blankenhorn,  M.  A.,  Arch.  Int.  Med.,  1916,  xviii, 289. 

10.  Hoppe-Seyler,   F.,  Handbuch  der  physiol.   u.   path-chem.  Analyse., 

Berlin,  8th  edition,  1909,  705;  Z.  physiol.  Chem.,  1881,  v,  1. 

11.  Huppert,  H.,  Arch.  Heilk.,  1864,  v,  236. 

12.  Inouye,  K.,  and  Ito,  H.,  Z.  physiol.  Chem.,  1908,  Ivii,  313. 

13.  Jolles,  A.,  Ber.  chem.  Ges.,  1908,  xli,  2766. 

14.  Kunkel,  A.,  Arch.  ges.  Physiol.,  1877,  xiv,  344. 

15.  Long,  J.  H.,  and  Gephart,  F.,  /.  Am.  Chem.  Soc.,  1908,  xxx,  1312. 

16.  Marshall,  E.  K.,  Jr.,  and  Davis,  D.  M.,  J.  Biol.  Chem.,  1914,  xviii,  60. 

17.  Neukomm,  J.,  Ann.  Chem.,  1860,  cxvi,  38. 

18.  Pfaff,  F.,  and  Balch,  A.  W.,  /.  Exp.  Med.,  1897,  ii,  49. 

19.  Socoloff,  N.,  Arch.  ges.  Physiol.,  1875,  xi,  166. 

20.  Spiro,  P.,  Arch.  Physiol.,  1880,  Suppl.  50. 

21.  Stadelmann,  E.,  Z.  Biol.,  1897,  xxxiv,  1. 

22.  von  Udranszky,  L.,  Z.  physiol.  Chem.,  1888,  xii,  355. 

23.  Ville,  J.,  and  Derrien,  E.,  Compt.  rend.  Soc.  biol.,  1909,  Ixvi,  175. 

24.  Van  Slyke,  D.  D.,  J.  Biol.  Chem.,  1913-14,  xvi,  127. 


THE  METABOLISM  OF  BILE  ACIDS 

II.    NORMAL  FLUCTUATIONS  IN  HEALTHY  BILE 
FISTULA  DOGS 


BY 

M.  G.  FOSTER,  C.  W.  HOOPER,  AND  G.  H.  WHIPPLE 


(FKOM  THE  GEORGE  WILLIAMS  HOOPER  FOUNDATION  FOR  MEDICAL 

RESEARCH,  UNIVERSITY  OF  CALIFORNIA  MEDICAL  SCHOOL, 

SAN  FRANCISCO) 


REPRINTED  FROM 

THE  JOURNAL  OF  BIOLOGICAL  CHEMISTRY 
VOL.  XXXVIII,  No.  2,  JUNE,  1919 


Reprinted  from  THE  JOURNAL  OF  BIOLOGICAL  CHEMISTRY,  Vol.  XXXVIII,  No.  2,  1919  ^  '  , ( 


THE  METABOLISM  OF  BILE  ACIDS. 

II.     NORMAL  FLUCTUATIONS  IN  HEALTHY  BILE  FISTULA  DOGS; 

BY  M.  G.  FOSTER,  C.  W.  HOOPER,  AND  G.  H.  WHIPPLE. 

(From  the  George  Williams  Hooper  Foundation  for  Medical  Research,  Uni- 
versity of  California  Medical  School,  San  Francisco.) 

(Received  for  publication,  April  28,  1919.) 

As  a  preliminary  to  any  series  of  bile  acid  experiments  it  is 
necessary  to  establish  the  normal  curve  of  excretion.  This  curve 
of  excretion  may  vary  with  the  condition  of  the  animal,  with  the 
diet  administered,  and  with  other  factors  which  may  not  be  sub- 
ject to  control.  Unless  otherwise  noted  the  dogs  used  in  these 
experiments  were  in  fine  physical  condition  as  shown  by  normal 
activity,  vigorous  appetite,  a  uniform  weight  curve,  and  normal 
blood  picture.  The  operative  procedure  and  general  routine 
care  of  these  animals  have  been  described  in  detail  in  an  earlier 
publication,  Hooper  and  Whipple  (1).  It  is  very  essential 
that  these  dogs  be  exercised,  fed,  set  up,  and  drained  regularly. 
Diet  regulation  is  very  important  arid  at  times  difficult.  Diar- 
rhea is  a  troublesome  feature  which  must  be  controlled,  and  this 
is  often  best  done  by  a  proper  admixture  of  kaolin  to  the  food. 
We  have  found  it  necessary  to  set  up  the  dogs  for  collection  of 
bile  at  least  30  minutes  before  the  actual  collection  is  started. 
This  assures  a  complete  drainage  of  the  thick  viscid  night  bile 
which  escapes  only  slowly  from  the  fistula.  The  presence  of 
this  concentrated  bile  in  the  first  collection  will  cause  high  read- 
ings and  introduce  an  error  unless  this  precaution  is  taken.  This 
explains  some  of  the  high  readings  in  the  first  2  hour  periods 
of  Tables  VIII  and  IX.  Unless  otherwise  stated,  dogs  were 
fed  exactly  2  hours  after  the  daily  collection  was  started  and 
again  after  the  period  of  exercise  following  the  collection. 
Weights  were  taken  in  the  morning  before  the  collection. 

367 


368  Metabolism  of  Bile  Acids.    II 


EXPERIMENTAL. 

The  method  of  chemical  analysis  has  been  controlled  and 
shown  to  be  reasonably  accurate.  The  method  of  bile  collection 
has  been  described  and  may  be  assumed  to  be  accurately  con- 
trolled. The  dogs  were  kept  in  a  room  in  which  they  were  under 
constant  supervision,  which  assured  an  accurate  and  complete 
bile  collection.  Occasionally  a  dog  may  be  restless  and  displace 
the  rubber  tube  in  the  fistula  allowing  the  escape  of  bile,  but  the 
binders  are  large  and  by  experience  carefully  fitted,  so  that  such 
accidents  are  rare.  When  there  was  loss  of  bile  the  material 
was  discarded  unless  the  loss  was  very  small  or  could  be  measured. 
Notes  are  made  of  any  deviations  from  the  uniform  routine  com- 
plete collection.  As  explained  in  another  place  we  feel  that  6 
or  8  hour  collections  are  more  satisfactory  than  the  longer  12 
to  24  hour  collections  used  by  Stadelmann  and  other  workers. 
The  longer  collections  are  more  trying  to  the  animal  and  usually 
cause  loss  of  weight,  appetite,  and  strength.  This  immediately 
introduces  the  factor  of  disease  with  its  many  unknown  vari- 
ables. We  feel  that  these  6  hour  collections  continued  over 
weeks  and  months  in  dogs  which  are  in  every  respect  healthy 
and  active  will  give  more  truthful  information  about  the  normal 
bile  acid  metabolism. 

It  is  to  be  noted  in  the  first  two  tables  (Tables  VIII  and  IX) 
that  the  dogs  were  not  drained  for  30  minutes  before  the  collec- 
tions were  begun.  The  first  2  hour  periods  show  a  bile  which 
is  more  concentrated  and  contains  more  bile  acids  than  does  the 
second  unit  period.  A  part  of  this  high  excretion  during  the 
first  2  hours  is  undoubtedly  to  be  explained  by  the  presence  of 
some  of  the  concentrated  night  bile  in  the  bile  passages,  not  com- 
pletely drained  off  before  collections  were  begun.  One  must 
keep  in  mind  the  normal  fluctuations  in  bile  acid  excretion  on  a 
mixed  diet  so  that  proper  care  may  be  exercised  in  the  interpre- 
tation of  the  fluctuations  noted  under  experimental  conditions. 
Fluctuations  in  bile  volume  are  at  tunes  startling  and  inexplicable. 
The  constitution  of  the  mixed  diet  may  account  for  some  of  the 
fluctuations  in  bile  volume.  It  has  been  pointed  out  elsewhere 
by  Whipple  and  Hooper  (4)  that  a  meat  diet  produces  a  thin, 
pale,  voluminous  bile  excretion,  poor  in  bile  pigments;  and  further 


Foster,  Hooper,  and  Whipple 


369 


TABLE  VIII. 

Bile  Acid  Excretion — 2  Hour  Periods — Mixed  Diet. 
Dog  18-23.     Simple  Bile  Fistula. 


Amino  nitro- 

Tauro- 

Date. 

Hour. 

Bile. 

gen. 

cholic 
acid 

Weight. 

Remarks. 

Per  cc. 

Out- 

output. 

of  bile. 

put. 

1918 

cc. 

ing. 

mg. 

mg. 

Ibs. 

Mar.  5 

1-2 

20 

0.374 

7.48 

Mixed  diet. 

3-4 

26 

0.231 

6.00 

5-6 

29 

0.158 

4.58 

1-6 

75 

18.06 

664 

32.0 

Mar.  6 

1-2 

17 

0.344 

5.85 

3-4 

26 

0.228 

5.93 

5-6 

21 

0.198 

4.16 

1-6 

64 

15.94 

585 

32.0 

Mar.  7 

1-2 

25 

0.253 

6.32 

Hb.  110  per  cent. 

3-4 

28 

0.170 

4.76 

R.  B.  C.  6,290,000. 

5-6 

32 

0.114 

3.65 

1-6 

85 

14.73 

541 

33.3 

Mar.  8 

1-2 

22 

0.264 

5.80 

3-4 

34 

0.190 

6.46 

5-6 

25 

0.191 

4.77 

1-6 

81 

17.03 

626 

34.0 

Mar.  11 

1-2 

34 

0.247 

,   8.40 

3-4 

32 

0.160 

5.11 

5-6 

25 

0.189 

4.72 

1-6 

91 

18.23 

670 

32.8 

Mar.   12 

1-2 

23 

0.253 

5.82 

3^ 

27 

0.148 

3.99 

5-6 

19 

0.153 

2.90 

1-6 

69 

12.71 

467 

34.0 

Mar.  13 

1-2 

28 

0.368 

10.30 

3-4 

29 

0.368 

10.67 

5-6 

25 

0.225 

5.63 

1-6 

82 

26.60 

978 

35.0 

Mar.  14 

1-2 

21 

0.315 

6.61 

3-4 

21 

0.286 

6.60 

5-6 

18 

0.300 

5.40 

1-6 

60 

18.61 

662 

35.5 

Mar.  15 

1-2 

34 

0.390 

13.26 

3^t 

22 

0.277 

6.09 

t 

5-6 

26 

0.223 

5.80 

1-6 

82 

25.15 

923 

34.8 

370 


Metabolism  of  Bile  Acids.    II 


TABLE  IX. 

Bile  Acid  Excretion — 2  Hour  Periods- — Mixed  Diet. 
Dog  17-151.    Simple  Bile  Fistula. 


Amino  nitro- 

Tauro- 

Date 

Hour. 

Bile. 

cholic 

Weight. 

Remarks. 

Per  cc. 

Out- 

output . 

of  bile. 

put. 

1918 

cc. 

mg. 

mg. 

mg. 

Ibs. 

Mar.  5 

1-2 

7 

0.828 

5.79 

3-4 

26 

0.675 

17.55 

5-6 

28 

0.329 

9.21 

1-6 

61 

32.55 

1,196 

42.5 

Mar.  6 

1-2 

11 

1.26 

13.86 

3-4 

23 

0.549 

12.62 

5-6 

14 

0.379 

5.30 

1-6 

48 

31.78 

1,167 

41.8 

Mar.  7 

1-2 

24 

0.409 

9.81 

Hb.  118  per  cent. 

3-4 

25 

0.267 

6.67 

R.  B.C.  6,350,000. 

5-6 

31 

0.213 

6.70 

1-6 

80 

23.18 

852 

42.5 

Mar.  8 

1-2 

13 

0.618 

8.03 

3-4 

6 

0.816 

4.89 

5-6 

13 

0.717 

9.32 

1-6 

32 

22.24 

818 

43.0 

Mar.  11 

1-2 

13 

0.437 

5.68 

3^ 

15 

0.278 

4.17 

5-6 

10 

0.101 

1.01 

1-6 

38 

10.86 

400 

41.5 

Mar.  12 

1-2 

13 

0.780 

10.14 

3-4 

13 

0.780 

10.14 

5-6 

4 

0.717 

2.86 

1-6 

30 

23.14 

850 

43.0 

Mar.  13 

1-2 

10 

1.00 

10.00 

3-4 

3 

0.691 

2.07 

5-6 

15 

0.663 

9.94 

1-6 

28 

22.01 

809 

42.5 

Mar.  14 

1-2 

32 

0.656 

20.99 

Ml 

5-6J 

8 

0.457 

3.65 

2nd  and  3rd  collec- 
tions  combined. 

1-6 

40 

24.64 

906 

43.5 

Mar.  15 

1-2 

13 

0.717 

9.32 

3^ 
5-6 

5\ 

4J 

0.654 

5.88 

2nd  and  3rd  collec- 
tions combined. 

1-6 

22 

15,20 

559 

44.0 

Foster,  Hooper,  and  Whipp]e 


371 


that  a  carbohydrate  diet  is  associated  with  a  thick,  scanty  bile 
excretion,  rich  in  bile  pigments.  The  mixed  diet  used  in  these 
experiments  consists  of  a  variable  mixture  of  kitchen  scraps  con- 
taining meat,  bones,  bread,  potato,  rice,  soup,  etc. 

Table  VIII  shows  the  variations  of  bile  salt  excretion  from 
day  to  day  in  2  hour  collections.  No  preliminary  drainage. 

Table  IX  shows  the  variation  in  bile  acid  excretion  from  day 
to  day  in  2  hour  collections.  No  preliminary  drainage. 

Hourly  Variations  in  Bile  Acid  Elimination. 

Dogs  were  set  up  for  8  and  9  hours  to  establish  the  hourly 
curve  of  daily  excretion  of  bile  acids.  Compare  Tables  X  and 
XI  with  Tables  VIII  and  IX  of  this  paper  and  subsequent  tables 
in  Paper  III.  These  experiments  are  characteristic  of  many 
others  .which  need  not  be  tabulated  at  this  time,  but  some  of 
these  supplementary  observations  will  be  given  in  later  papers 
to  prove  other  points. 

TABLE  x. 

Bile  Acid  Excretion- — Hour  Periods — Mixed  Diet. 
Dog  18-137.    Simple  Bile  Fistula  and  Splenectomy. 


Hour. 

Volume  . 

Amino  nitnxren  . 

Tauro- 
cholic 
acid  per 
hour. 

Remarks. 

Per  cc.of 
bile. 

Per 
hour. 

cc. 

mg. 

mg. 

mg. 

I        . 

9.0 

0.317 

2.85 

104 

October  1. 

2  and  3 

20.0 

0.302 

3.00 

110* 

4 

9.0 

0.274 

2.47 

90 

5 

12.0 

0.230 

2.76 

101 

End  of  5th  hour  fed  mixed  diet. 

6 

10.5 

0.230 

2.41 

88 

7  and  8 

22.0 

0.216 

2.37 

87* 

Hb.  90  per  cent. 

R.  B.  C.  3,830,000. 

9 

10.5 

0.230 

2.41 

88 

Weight  24.0  Ibs. 

*  Average  of  2  hours. 

Table  X  is  quite  typical  of  a  group  of  experiments  and  shows 
a  nearly  uniform  hourly  elimination  of  bile  acids.  There  is  a 
general  tendency  for  the  bile  acid  excretion  curve  to  fall  slightly 
in  the  afternoon  in  spite  of  a  midday  feeding  and  careful  pre- 
liminary drainage  of  the  concentrated  night  bile  before  begin- 
ning the  experiment. 


372 


Metabolism  of  Bile  Acids.    II 


TABLE  XI. 

Bile  Acid  Excretion — Hour  Periods — Meat  Diet. 
Dog  17-84'     Bile  Fistula  and  Splenectomy. 


Hour. 

Volume. 

Amino  nitrogen. 

Tauro- 
cholic  acid 
per  hour. 

Remarks. 

Per  cc.  of 
bile. 

Per  hour. 

cc. 

mg. 

mg. 

mg. 

December  11. 

1 
2 

3.8 
9.1 

0.686 
0.438 

2.61 
3.98 

96 

147 

Fed  500  gm.  of  meat. 
"    300    "      "      " 

3 

9.1 

0.351 

3.19 

118 

Weight  29.  8  Ibs. 

4 

6.4 

0.614 

3.93 

143 

5 

4.1 

0.614 

2.52 

92 

6 

5.7 

0.614 

3.50 

129 

7 

5.1 

0.658 

3.36 

124 

8 

3.9 

0.731 

2.85 

106 

9 

6.0 

0.686 

4.10 

150 

TABLE  Xl-a. 


Bile  Acid  Excretion — 2  Hour  Periods — Fasting  and  Meat  Diet. 
Dog  17-34.    Bile  Fistula  and  Splenectomy. 


Amino 

C5  C^I 

• 

—    00 

nitrogen. 

'^•a-» 

3     «H 

Hour. 

1 

8-a* 

•a£ 

Remarks. 

3 

•a 

> 

Per  cc. 
of  bile. 

In 
2  hours. 

tL  .—    3 
3  «  0 

£*' 

£  .C 

s"* 

cc. 

mg. 

mg. 

mg. 

mg. 

February  13. 

1-2 

22 

0.440 

9.68 

355 

15.3 

Fasting.    Mixed  diet  day  before 

fasting. 

3-4 

20 

0.332 

6.64 

242 

'13.2 

5-6 

20 

0.318 

6.36 

235 

13.9 

Weight  31.  8  Ibs. 

7-8 

18 

0.303 

5.45 

201 

13.1 

9-10 

19 

0.318 

6.04 

220 

7.0 

February  19  to  26 — Rice,  potato,  and  milk  diet. 


February  27. 

1-2 

21 

0.189 

3.96 

146 

11.0 

Meat  250  gm.  at  beginning. 

3-4 

16 

0.132 

2.11 

77 

8.9 

5-6 

20.5 

0.161 

3.30 

121 

12.4 

Weight  27.5  Ibs. 

7-8 

14 

0.247 

3.46 

129 

11.2 

9-10 

13 

0.261 

3.39 

124 

11.8 

Foster,  Hooper,  and  Whipple 


373 


Tables  XI  and  Xl-a  show  slight  fluctuations  in  bile  acid  out- 
put after  feeding,  but  we  do  not  attach  any  significance  to  this 
reaction.  It  will  be  noted  (Table  Xl-a)  that  the  level  of  bile 

TABLE  XII. 

Bile  Acid  Excretion  Not  Influenced  by  "Bile  Exclusion."* 
Dog  18-23.    Simple  Bile  Fistula. 


Amino 

CO 
U  t£          1        *-l 

nitrogen. 

'o  a 

.3  . 

Date. 

1 
1 

In  Ice. 

of  bile. 

In 
6  hours 

|a|    11 
5  ss  ,|a 

1 
8 

Remarks. 

1918 

cc. 

mg. 

mg. 

mg.         mg. 

Ibs. 

June    3 

62 

0.326 

20.20 

741      6.5 

30.2 

No  bile  exclusion. 

"       4 

60 

0.409 

24.54 

902 

13.6 

31.2 

Mixed  diet. 

"       5 

61 

0.422 

25.74 

932 

20.9 

31.2 

"       6 

82 

0.371 

30.42 

1,115 

11.2 

"       7 

75 

0.191 

14.33 

526 

24.8 

32.2 

"       8 

58 

0.284 

16.57 

608 

16.2 

32.0 

Hb.  127  per  cent. 

R.  B.  C.  6,665,000. 

"     10 

75 

0.224 

16.80 

618 

22.2 

30.2 

* 

"     11 

77 

0.289 

22.25 

817 

28.7 

31.7 

Average  

21.4 

782 

No  bile  exclusion. 

June  12 

80 

0.199 

15.92 

584 

23.7 

32.0 

Absolute  bile  exclusion. 

"    13 

78 

Lost. 

16.3 

32.0 

Mixed  diet. 

"     14 

96 

0.346 

33.22 

1,215 

20.0 

32.0 

"     15 

72 

0.248 

17.86 

656 

30.2 

16 

Dog  set  up  and  drained 

2  hrs. 

"    17 

66 

0.180 

11.88 

436 

24.1 

30.5 

"     18 

83 

0.238 

19.75 

725 

23.3 

32.0 

- 

Average 

19.72 

723 

Complete  bile  exclusion. 

*  "Bile  exclusion"  means  total  inability  of  the  dog  to  lick  any  bile  from 
the  fistula  at  any  time.      This  is  effected  by  means  of  a  thick  gauze  pad  and 


large  binder. 


This 


acid  excretion  is  influenced  by  the  diet  of  the  previous  day. 
point  will  be  taken  up  again. 

This  method  makes  it  possible  to  follow  the  hourly  fluctuations 
in  the  bile  acid  output  and  to  establish  for  the  first  time  the  actual 
hourly  curve  of  bile  acid  elimination  from  hour  to  hour. 


374 


Metabolism  of  Bile  Acids.    II 


TABLE  XIII. 

Bile  Acid  Excretion  Not  Influenced  by  "Bile  Exclusion.' 
Dog  18-23.    Simple  Bile  Fistula. 


Amino 

CO 

£3 

•pv_  j  , 

; 

nitrogen. 

la   . 

i« 

JJate. 

i 

2 

Inl 
cc.  of 
bile. 

In  6 

hours. 

jll 

IS 
p 

PI 

Jd 

Remarks. 

1918 

cc. 

mg. 

mg. 

mg. 

mg. 

Ibs. 

No  bile  exclusion. 

May  21 

57 

0.223 

12.72 

467 

12.9 

30.75 

Diet  300  gm.  of  cracker  meal, 

65  gm.  of  meat. 

"    22 

52 

0.255 

13.26 

487 

10.8 

30.75 

Diet  300  gm.  of  cracker  meal, 

65  gm.  of  meat. 

"    23 

73 

0.323 

23.58 

866 

12.9 

30.50 

Diet  300  gm.  of  cracker  meal, 

65  gm.  of  meat. 

"    24 

61 

0.253 

15.44 

567 

12.9 

30.25 

Diet  300  gm.  of  cracker  meal, 

65  gm.  of  meat. 

"  25-26 

30.25 

Mixed  diet. 

"    27 

66 

30.25 

300  gm.  of  cracker  meal,  65 

gm.  of  meat. 

"    28 

72 

0.221 

15.90 

574 

7.5 

30.50 

300  gm.  of  cracker  meal,  65 

gm.  of  meat. 

"    29 

82 

0.334 

27.40 

1,005 

11.2 

30.25 

300  gm.  of  cracker  meal,  65 

gm  of  meat. 

"    30 

300  gm.  of  cracker  meal,  65 

gm.  of  meat. 

"    31 

72 

0.210 

15.12 

552 

10.0 

30.00 

Average  

17.6 

645 

No  bile  exclusion. 

Absolute  bile  exclusion. 

June  19 

77 

0.252 

19.40 

712 

18.8 

32.50 

300  gm.  of  cracker  meal,  65 

gm.  of  meat. 

"    20 

78 

0.210 

16.38 

602 

23.7 

32.00 

300  gm.  of  cracker  meal,  65 

gm.  of  meat. 

"    21 

59 

0.294 

17.35 

637 

19.8 

31.50 

300  gm.  of  cracker  meal,  65 

gm.  of  meat. 

"    22 

28 

0.476 

13.32 

489 

23.4 

31.00 

300  gm.  of  cracker  meal,  65 

gm.  of  meat. 

Average 

16.6 

610 

Complete  bile  exclusion. 



The  question  of  "bile  exclusion"  is  a  very  important  one  for 
this  entire  series  of  experiments.  The  question  resolves  itself 
into  the  following:  Does  a  bile  fistula  dog  lick  enough  bile  from 


Foster,  Hooper,  and  Whipple  375 

its  fistula  during  the  night  to  influence  in  any  way  the  daily  ex- 
cretion of  bile  or  bile  acids?  According  to  Stadelmann  (3)  in 
his  experiments  this  "bile  exclusion"  is  necessary.  The  ex- 
clusion of  any  possible  ingestion  of  the  dog's  own  bile  in  his  experi- 
ments would  cause  a  decrease  of  about  f  the  total  bile  acid  ex- 
cretion. Because  of  our  respect  for  Stadelmann's  work  we  felt 
that  it  was  necessary  to  control  this  point  beyond  the  peradven- 
ture  of  a  doubt.  We  submit  a  sufficient  number  of  experiments 
in  Paper  V  to  prove  that  under  the  conditions  of  our  experiments 
"bile  exclusion"  does  not  influence  the  output  of  bile  acids.  The 
reasons  for  this  are  discussed  more  in  detail  in  that  paper.  These 
experiments  (Tables  XII  and  XIII)  make  the  same  point  and  are 
sufficient  to  inform  the  reader  that  this  important  factor  has  been 
properly  controlled  in  experiments  that  follow.  Absolute  "bile 
exclusion"  does  not  affect  the  output  of  bile  acids  on  either  a 
mixed  diet  or  a  known  diet.  The  bile  salt  excretion  can  be  partly 
controlled  by  diet,  as  will  be  shown  in  another  paper.  A  diet 
rich  in  meat  protein  increases  the  output  and  a  diet  poor  in  meat 
protein  reduces  the  excretion. 

Effect  of  Bile  by  Mouth  on  the  Following  Day's  Excretion. 

In  order  to  find  out  if  bile  by  mouth  in  moderate  amounts 
affected  the  following  day's  excretion  several  dogs  were  given 
bile  at  night  and  the  bile  acid  excretion  followed. 

Table  XIV  shows  that  bile  feeding  in  the  late  afternoon  does 
not  influence  the  following  day's  excretion  of  bile  acids.  Many 
other  experiments  have  been  performed  with  identical  results. 
It  is  apparent  from  experiments  tabulated  in  Paper  III  that  the 
greater  part  (about  80  per  cent)  of  the  bile  acids  ingested  as  bile 
will  appear  in  the  bile  fistula  bile  within  4  hours.  We  may  as- 
sume that  only  bile  ingested  during  the  early  morning  hours  (5 
to  8  a.  m.)  influences  the  bile  collections  in  our  experiments. 
There  is  no  clinical  evidence  that  the  dogs  lick  any  bile  from  their 
fistulas  during  this  period  and  the  experimental  data  confirm 
this  point. 


376 


Metabolism  of  Bile  Acids.  II 


TABLE  XIV. 

Bile  Feeding  at  Night — Mixed  Diet. 
Simple  Bile  Fistula  with  Splenectomy . 


Amino 

O  CO 

nitrogen. 

Dog 

Date. 

OJ 

|.sg- 

^ 

Remarks. 

No. 

£ 
J3 

Inl 
cc.  of 
bile. 

In  6 
hours. 

3'5  0 

£*" 

if 

17-181 

Average    previous- 

10  days. 

10.60 

389 

1918 

CC. 

mg. 

mg. 

mg. 

Ibs. 

July    29 

4  p.m.  given  58  cc.  of^bile  by 

stomach  tube. 

"      30 

36 

0.267 

9.61 

352 

25.50 

5  p.m.  given  60  cc.  of  bile  by 

stomach  tube. 

"       31 

25 

0.418 

10.45 

384 

26.25 

5  p.m.  given  60  cc.  of  bile  by 

stomach  tube. 

Aug.     1 

38 

0.285 

10.82 

397 

25.00 

No  bile  given. 

"        2 

37 

0.244 

9.03 

332 

25.25 

Hb.  100  per  cent. 

R.  B.  C.  4,500,000. 

18-137 

Average    previous 

10  days. 

14.9 

547 

July   29 

22.75 

4  p.m.  given  58  cc.  of  bile  by 

stomach  tube. 

"      30 

50 

0.310 

15.50 

569 

23.50 

4  p.m.  given  60  cc.  of  bile  by 

stomach  tube. 

"      31 

42 

0.401 

16.80 

617 

24.0 

5  p.m.  given  60  cc.  of  bile  by 

stomach  tube. 

Aug.     1 

43 

0.395 

16.55 

608 

23.5 

No  bile  given. 

"        2 

30 

0.145 

4.35 

159 

22.75 

Hb.  100  per  cent. 

R.  B.  C.  4,425,000. 

DISCUSSION. 

Loeb  (2)  stated  that  the  sulfur  and  nitrogen  excretion  in 
the  bile  is  higher  during  the  first  4  hours  than  during  the  second 
4  hours  after  eating.  From  the  tables  given  in  this  paper  it  is 
clear  that  the  bile  acid  excretion  is  greater  during  the  early  part 
of  the  day  than  later  in  the  afternoon.  There  is  always  a  ten- 
dency to  fall  off  during  the  5th  and  6th  hours  of  the  collection 
in  spite  of  the  ingestion  of  food.  But  in  an  individual  the  amount 


Foster,  Hooper,  and  Whipple  377 

excreted  throughout  the  day  is  fairly  uniform  provided  a  fore- 
period  of  30  minutes  or  longer  for  drainage  of  the  bile  fistula 
has  been  a  part  of  the  routine  preceding  the  usual  6  hour  collection. 
It  is  evident  that  the  amount  of  bile  which  these  dogs  normally 
lick  from  their  fistulas  during  the  hours  they  spend  in  their  cages 
is  insufficient  to  cause  any  variation  in  the  following  day's  out- 
put (Tables  XII  and  XIII).  The  only  time  our  dogs  are  prone 
to  lick  their  fistulas  is  just  after  the  completion  of  the  daily  col- 
lection. But  Table  XIV  shows  that  moderate  amounts  of  bile 
at  this  time  are  excreted  before  the  following  day's  collection  is 
made,  and  do  not  affect  the  determination  in  any  way. 

SUMMARY. 

Bile  acid  excretion  in  a  healthy  bile  fistula  dog  given  a  mixed 
diet  will  show  great  variations  from  day  to  day. 

The  amount  of  bile  acid  excreted  is. usually  somewhat  higher 
in  the  morning  than  in  the  afternoon.  This  holds  good  even  after 
complete  drainage  (J  hour)  of  the  concentrated  night  bile,  and 
in  spite  of  liberal  feeding  2  hours  after  collections  are  begun. 

The  amount  of  bile  acid  excreted  hour  by  hour  during  any 
given  day  is  fairly  uniform. 

The  amount  of  bile  which  a  dog  may  lick  from  its  fistula  during 
the  afternoon  and  night  resting  period  is  not  sufficient  in  our 
experiments  to  cause  any  demonstrable  variation  in  the  following 
day's  excretion. 

Moderate  amounts  of  bile  given  by  stomach  in  the  late  after- 
noon do  not  influence  the  following  day's  excretion  of  bile  acids. 

BIBLIOGRAPHY. 

1.  Hooper,  C.  W.,  and  Whipple,  G.  H.,  Am.  J.  PhysioL,  1916,  xl,  332. 

2.  Loeb,  A.,  Z.  BioL,  1911,  Iv,  167. 

3.  Stadelmann,  E.,  Z.  physiol.  Chem.,  1897,  xxxiv,  1. 

4.  Whipple,  G.  H.,  and  Hooper,  C.  W.,  Am.  J.  PhysioL,  1916,  xl,  349. 


THE  JOURNAL   OF  BIOLOGICAL  CHEMISTRY,    VOL.  XXXVIII,  NO.  2 


THE  WAVERLY  PHE 

BALTIMORE.  U.  0.  A. 


THE  METABOLISM  OF  BILE  ACIDS 

III.    ADMINISTRATION  BY  STOMACH  OF  BILE,  BILE 

ACIDS,   TAURINE,  AND  CHOLIC  ACID  TO 

SHOW  THE  INFLUENCE  UPON  BILE 

ACID  ELIMINATION 


BY 

M.  G.  FOSTER,  C.  W.  HOOPER,  AND  G.  H.  WHIPPLE 


(FROM  THE  GEORGE  WILLIAMS  HOOPER  FOUNDATION  FOR  MEDICAL 

RESEARCH,  UNIVERSITY  OF  CALIFORNIA  MEDICAL  SCHOOL, 

SAN  FRANCISCO) 


REPRINTED  FROM 

THE  JOURNAL  OF  BIOLOGICAL  CHEMISTRY 
VOL.  XXXVIII,  No.  2,  JUNE,  1919 


Reprinted  from  THE  JOURNAL  OF  BIOLOGICAL  CHEMISTRY,  Vol.  XXXVIII,  No.  2,  1919. 


THE  METABOLISM  OF  BILE  ACIDS. 

III.    ADMINISTRATION  BY  STOMACH  OF  BILE,  BILE  ACIDS,  TAUR- 

INE,  AND  CHOLIC  ACID  TO  SHOW  THE  INFLUENCE 

UPON  BILE  ACID  ELIMINATION. 

BY  M.  G.  FOSTER,  C.  W.  HOOPER,  AND  G.  H.  WHIPPLE. 

(From  the  George  Williams  Hooper  Foundation  for  Medical  Research,  Uni- 
versity of  California  Medical  School,  San  Francisco.) 

(Received  for  publication,  April  28,  1919.) 

The  cholagogue  action  of  whole  bile  given  by  stomach  is  a  well 
known  fact.  It  has  been  established  by  many  experiments  that 
the  curve  of  bile  volume  excretion  does  not  necessarily  parallel  the 
curve  of  excretion  of  bile  pigments  (Whipple  and  Hooper,  9).  In 
other  words  it  is  possible  to-  stimulate  a  free  flow  of  bile  which  is 
poor  in  bile  pigments,  so  much  so  that  the  total  bile  pigment  out- 
put may  fall  to  half  normal  during  a  period  of  active  cholagogue 
excretion  with  twice  normal  output  of  bile.  Hooper  (3)  has 
pointed  out  several  factors  which  may  contribute  to  this  reaction. 
It  is  well  to  recall  that  taurocholic  acid  given  by  mouth  has  a 
marked  cholagogue  action — experiments  given  below  add  more 
data  to  establish  this  statement.  An  ether  extract  of  dried  bile, 
which  of  course  contains  a  mixture  of  substances,  has  no  chola- 
gogue action,  but  inhibits  the  excretion  of  bile  pigments.  It  is  of 
considerable  interest  to  know  that  certain  substances  can  stimu- 
late or  inhibit  the  total  bile  excretion  while  influencing  the  output 
of  the  various  bile  constituents  in  the  same  or  opposite  direction. 

In  1875  Socoloff  (7)  injected  glycocholic  acid  into  a  dog  and 
found  an  increased  excretion  but  no  increase  in  the  per  cent  of  bile 
acids.  But  his  method  was  questionable  and  he  used  only  one 
dog.  Rutherford  and  Vignal  (5)  injected  bile  acids  into  the 
jejunum  and  found  an  increased  output  of  bile  acids,  but  they  do 
not  mention  how  the  bile  acids  were  estimated.  To  Schiff  (6)  is 
given  the  credit  for  having  established  the  fact  that  there  is  a 
reabsorption  of  bile  acid  and  this  is  often  termed  the  "circulation 

379 


380  Metabolism  of  Bile  Acids.     Ill 

of  the  bile."  This  term  "circulation  of  the  bile"  is  used  loosely 
by  some  writers  and  is  at  times  misquoted  to  indicate  a  circulation 
of  other  of  the  substances  found  in  the  bile,  for  example,  bile  pig- 
ments. We  have  definite  proof  that  bile  pigments  are  not  ab- 
sorbed from  the  intestine  (Hooper  and  Whipple,  4).  At  present 
we  have  little  if  any  accurate  knowledge  about  the  many  other 
substances  present  in  fresh  bile — they  may  be  absorbed  or  not. 

In  giving  dog's  bile  by  mouth,  Stadelmann  (8)  found  that  with  doses  of 
2.0  or  2.5  gm.  of  bile  salt,  the  cholagogue  action  lasted  up  to  24  or  36  hours, 
but  the  bile  salt  was  excreted  within  24  hours,  usually  within  10  hours. 
With  1.5  gm.  of  pure  sodium  glycocholate  the  whole  amount  was  excreted 
within  12  hours.  The  greatest  cholagogue  action  was  during  the  first  6 
hours.  From  then  on  it  began  to  abate  and  was  over  within  the  next  12 
hours.  The  bile  salts  may  have  been  excreted  in  less  than  10  to  12  hours, 
for  Stadelmann  performed  bile  salt  analyses  only  upon  these  large  col- 
lections. 

With  doses  of  3  to  5  gm.  morning  and  evening  the  salts  and  volume 
were  tremendously  increased  but  the  excretion  was  not  directly  propor- 
tional to  the  amounts  given.  5  gm.  twice  a  day  for  3  successive  days  kept 
both  the  volume  and  salts  above  normal  for  more  than  8  days,  even  though 
they  were  continually  decreasing.  These  experiments  of  Stadelmann's 
are  in  harmony  with  those  tabulated  below,  and  supplement  our  experi- 
mental data. 

One  is  not  surprised  to  observe  in  the  experiments  given  below 
that  the  curves  of  whole  bile  excretion  and  bile  acid  excretion  may 
run  parallel  after  the  oral  administration  of  fresh  dog's  bile — 
moreover,  on  the  contrary,  that  these  curves  may  be  widely  dis- 
sociated. A  moderate  dose  of  whole  dog's  bile  given  by  stomach 
will  cause  a  distinct  cholagogue  action  and  a  parallel  increase  in 
bile  acids.  With  the  fall  in  bile  volume  after  3  to  5  hours  there  is 
a  fall  in  the  bile  acid  output.  It  is  possible  to  give  sugar  with  con- 
centrated bile  by  stomach  tube  and  completely  inhibit  the  chola- 
gogue action  while  a  great  rise  in  output  of  bile  acids  is  taking 
place.  We  believe  this  procedure  gives  a  maximum  concentration 
of  bile  acids  in  dog's  bile,  as  high  even  as  7  to  9  per  cent  by  weight. 
Perhaps  this  represents  the  maximum  power  of  the  liver  cells  to 
concentrate  bile  acids  in  whole  bile,  at  least  under  the  conditions 
of  the  experiment. 


Foster,  Hooper,  and  Whipple 


381 


EXPERIMENTAL. 

The  first  four  tables  (Tables  XV  to  XVIII)  are  to  be  taken  as  a 
unit  because  they  illustrate  the  uniformity  of  reaction  which  fol- 
lows the  ingestion  of  moderate  amounts  of  fresh  dog's  bile.  The 
taurocholic  acid  content  varies  from  0.63  to  1.83  gm.  in  any  given 
single  dose.  The  hourly  curve  of  bile  acid  excretion  is  remarkably 

TABLE  xv. 

Bile  Feeding. 
Dog  17-181.    Bile  Fistula  and  Splenectomy. 


Hour. 

Volume. 

Amino  nitrogen. 

Tauro- 
cholic acid 
in  1  hour. 

Remarks. 

Per  cc.  of 
bile. 

In  1  hour. 

cc. 

mg. 

mg. 

mg. 

August  27. 

1 

8.0 

0.319 

2.55 

93 

End  of  1st  hour  given  50  cc. 

of    whole    dog's    bile    con- 

taining 752  mg.   of    tauro- 

cholic acid. 

2 

18.5 

0.667 

12.30 

452 

3 

11.0 

0.662 

7.28 

267 

4 

7.0 

0.596 

4.17 

153 

5 

6.5 

0.333 

2.16 

79 

End   of  5th  hour  fed  mixed 

diet. 

6 

7.0 

0.222 

1.55 

56 

7' 

10.5 

0.222 

2.33 

85 

8 

10.0 

0.210 

2.10 

77 

9 

9.0 

0.207 

1.86 

68 

Hb.  100  per  cent. 

R.  B.  C.  4,810,000. 

10 

10.0 

0.221 

2.21 

81 

Weight  25.3  Ibs. 

constant  and  usually  shows  that  the  largest  elimination  takes 
place  during  the  first  3  hours  after  ingestion.  Some  experiments 
show  the  maximum  elimination  of  bile  acids  during  the  first  hour, 
again  during  the  second  or  third  hour.  The  bile  acid  concentra- 
tion per  cc.  of  the  bile  eliminated  usually  closely  parallels  the  curve 
of  total  excretion.  The  cholagogue  action  is  marked  with  the 
larger  doses  but  is  practically  absent  following  the  smallest  dose. 

Table  XV  shows  that  about  90  per  cent  of  bile  acid  in  whole  bile 
given  by  mouth  is  excreted  within  3  hours. 


382 


Metabolism  of  Bile  Acids.     Ill 


TABLE  XVI. 

Bile  Feeding. 
Dog  18-137.    Bile  Fistula  and  Splenectomy. 


Hour. 

Volume. 

Amino  nitrogen. 

Tauro- 
cholic  acid 
inl  hour. 

Remarks. 

Per  cc.  of 
bile. 

In  1  hour. 

cc. 

mg. 

mg. 

mg. 

September  5. 

1 

10.0 

0.442 

4.42 

162 

2 

7.5 

0.313 

2.35 

86 

End   of  2nd   hour  75    cc.    of 

bile  containing  628  mg.  of 

taurocholic  acid. 

3 

11.0 

0.470 

5.17 

189 

4 

9.0 

0.666 

6.00 

220 

5 

8.5 

0.696 

5.92 

217 

End  of  5th  hour  fed  mixed 

diet. 

6 

7.0 

0.464 

3.25 

119 

7 

10.0 

0.275 

2.75 

101 

8 

8.0 

0.261 

2.25 

82 

Hb.  120  per  cent. 

R.  B.  C.  5,060,000. 

9 

8.0 

0.248 

1.98 

72 

Weight  22.25  Ibs. 

10 

7.0 

0.221 

1.55 

56 

TABLE  XVII. 


Bile  Feeding. 
Dog  18-23.    Simple  Bile  Fistula. 


Hour. 

Volume. 

Amino  nitrogen. 

Tauro- 
cholic 
acid. 

Remarks. 

Per  cc.  of 
bile. 

In  1  hour. 

cc. 

mg. 

mg. 

mg. 

August  6. 

1 

7.5 

0.364 

2.73 

100 

End  1st  hour  given  100  cc.  of 

bile  containing  1.825  gm.  of 

taurocholic  acid. 

2 

22.0 

0.757 

16.65 

612 

3 

17.0 

0.980 

16.65 

612 

4 

20.5 

0.925 

18.95 

696 

5 

13.5 

0.582 

7.86 

288 

End  of  5th  hour  fed  mixed 

diet. 

6 

11.0 

0.373 

4.10 

150 

7 

10.5 

0.276 

2.90 

106 

Hb.  120  per  cent. 

R.  B.  C.  6,375,000. 

8 

9.0 

0.345 

3.10 

114 

Weight  31  .5  Ibs. 

TABLE  XVIII. 

Bile  Feeding. 
Dog  18-137.    Bile  Fistula  and  Splenectomy. 


Hour. 

Volume. 

Amino  nitrogen. 

Tauro- 
cholic acid 
in  1  hour. 

Remarks. 

Per  cc.  of 
bile. 

In  1  hour. 

cc. 

mg. 

mg  . 

mg. 

September  24. 

1 

8.5 

0.530 

4.51 

165 

2 

11.0 

0.430 

4.73 

173 

End  of  2nd  hour  100  cc.  of 

bile  containing  951  mg.  of 

taurocholic  acid. 

3 

18.0 

0.788 

14.18 

520 

4 

16.0 

0.845 

13.51 

499 

5 

10.0 

0.516 

5.16 

189 

End   of  5th  hour  fed  mixed 

diet. 

6 

10.5 

0.508 

5.33 

195 

7 

11.0 

0.326 

3.58 

131 

8 

11.0 

0.312 

3.43 

126 

Hb.  90  per  cent. 

R.  B.  C.  3,830,000. 

9 

10.0 

0.260 

2.60 

95 

Weight  23.3  Ibs. 

TABLE  XIX. 

Concentrated  Bile  Feeding. 
Dog  17-34.     Bile  Fistula  and  Splenectomy. 


TT^,,, 

Amino  nitrogen. 

Tauro- 

xlour. 

Volume. 

Per  cc.  of 
bile. 

In  1  hour. 

cholic  acid 
in  1  hour. 

Remarks. 

cc. 

mg. 

mg. 

mg. 

October  15. 

i 

7.5 

0.546 

4.09 

150 

2 

6.4 

0.445 

2.85 

104 

End  2nd  hour  170  cc.  of  con- 

centrated     bile      solution* 

containing     11.50     gm.     of 

taurocholic   acid    given   by 

stomach  tube. 

3 

21.0 

0.968 

20.31 

745 

4 

26.0 

1.430 

37.20 

1,365 

Vomited  about  200  cc.  of  fluid. 

5 

29.0 

1.360 

39.44 

1,445 

End  5th  hour  fed  mixed  diet. 

6 

34.3 

1.220 

41.88 

1,535 

7 

54.0 

1.000 

54.00 

1,980 

8 

18.0 

0.657 

11.82 

433 

Hb.  108  per  cent. 

R.  B.  C.  5,700,000. 

9 

12.0 

0.299 

3.59 

132 

Weight  31.5  Ibs. 

Oct.  16 

60. 

0.313 

18.80 

690 

Usual  6  hour  collection. 

Oct.  17 

64. 

0.328 

21.00 

752 

Usual  6  hour  collection. 

Oct.  18 

56. 

0.300 

16.80 

616 

Usual  6  hour  collection. 

*  Solution  prepared  by  evaporating  bile   to  dryness,  extracting  with 
water,  and  centrifuging.     The  qlear  supernatant  fluid  is  this  bile  solution. 

383 


384 


Metabolism  of  Bile  Acids.     Ill 


Table  XVI  shows  that  about  60  per  cent  of  bile  acid  adminis- 
tered by  mouth  was  excreted  within  4  hours.  Note  little  if  any 
cholagogue  action. 

Table  XVII  shows  that  about  90  per  cent  of  bile  acid  adminis- 
tered by  mouth  was  excreted  in  4  hours. 

Table  XVIII  shows  that  about  85  per  cent  of  bile  acid  given  in 
bile  by  mouth  is  excreted  within  4  hours. 

TABLE  xx. 

Concentrated  Bile  Feeding. 
Dog  18-137.    Simple  Bile  Fistula  and  Splenectomy. 


Hour. 

Volume. 

Aminq  nitrogen. 

Tauro- 
cholic  acid 
in  1  hour. 

Remarks. 

Perec,  of 
bile. 

In  1  hour. 

cc. 

mg. 

mg. 

mg. 

September  18. 

1 

7.5 

0.187 

1.40 

51 

2 

12.0 

0.330 

3.96 

145 

End  2nd  hour  370  cc.  of  bile 

solution  f    containing    11.60 

gm.     of     taurocholic     acid 

given  by  stomach  tube. 

3 

16.5 

0.729 

12.00 

440 

Vomited  60  cc. 

4 

20.0 

1.111 

22.22 

816 

5 

16.0 

1.200 

19.20 

704 

End  5th  hour  fed  mixed  diet. 

.     6 

14.0 

1.010 

14.15 

519 

Hb.  120  per  cent. 

R.  B.  C.  5,060,000. 

7,  8,  and 

45.0 

0.750 

11.24* 

413* 

Weight  23.5  Ibs. 

9 

*  Average  of  3  hours. 

t  Solution  prepared  by  evaporating  bile  'to  dryness,  extracting  with 
water,  and  centrifuging.  The  clear  supernatant  fluid  is  this  bile  solution. 

Tables  XIX  and  XX  show  remarkably  well  the  intense  reaction 
which  may  follow  large  doses  of  taurocholic  acid  by  mouth. 
Whole  bile  was  not  given,  but  a  crude  watery  extract  of  dried 
dog's  bile  which  is  rich  in  taurocholic  acid.  Vomiting  occurred 
and  the  amount  regurgitated  is  not  known. 

The  cholagogue  action  is  noted  immediately  and  is  sustained 
many  hours  and  even  days  (Table  XIX).  The  concentration  of 
bile  acids  per  cc.  of  bile  is  much  above  normal  and  the  actual  out- 
put of  almost  2  gm.  per  hour  is  reached  in  one  experiment.  Large 


Foster,  Hooper,  and  Whipple 


385 


amounts  of  bile  acids  seem  to  cause  no  ill  effects,  immediate  or 
delayed,  except  some  nausea  and  vomiting.  What  effect  if  any  is 
produced  in  the  general  body  metabolism  is  unknown. 

Table  XIX  shows  that  bile  acid  fed  by  mouth  in  high  concentra- 
tion causes  a  great  increase  in  bile  acid  output  for  6  hours,  and  is  a 

TABLE  XXI. 

Concentrated  Bile  Feeding  Plus  Sugar. 
Dog  15-22.     Simple  Bile  Fistula. 


Amino  nitrogen. 

Tauro- 

Hour. 

Volume. 

Per  cc.  of 
bile. 

In  1  hour. 

cholic  acid 
in  1  hour. 

Remarks. 

cc. 

mg. 

mg. 

mg. 

September  26. 

1 

21.5 

0.675 

14.50 

532 

2 

22.0 

0.380 

8.36 

301 

End  of  2nd  hour  125  cc.  of 

bile  solution*  containing 

8.770   gm.   of   taurocholic 

acid  —  100   gm.    of   sugar- 

water.    Little  vomiting. 

3 

15.0 

1.54 

23.10 

848 

4 

9.5 

1.93 

18.35 

673 

5 

14.5 

2.31 

33.50 

1,230 

End  of  5th  hour  fed  mixed 

diet. 

6 

15.5 

2.35 

36.45 

1,340 

7 

15.0 

2.26 

33.90 

1,245 

8 

11.0 

1.80 

19.80 

727 

Hb.  110  per  cent. 

R.  B.  C.  5,280,000. 

9 

8.5 

1.77 

15.05 

552 

Weight  32.8  Ibs. 

Sept.  27. 

37.0 

1.19 

44.00 

1,615 

Usual  6  hour  collection. 

Sept.  30. 

12.0 

1.18 

14.10 

518 

Usual  6  hour  collection. 

*  Bile  evaporated  to  dryness,  the  residue  extracted  with  water,  and 
centrifuged.  The  clear  supernatant  fluid  is  this  bile  solution. 

cholagogue  for  a  much  longer  period.  About  65  per  cent  was 
excreted  in  6  hours. 

Table  XX  shows  that  concentrated  bile  acid  by  mouth  may 
cause  a  long  delayed  bile  acid  excretion. 

Table  XXI  shows  that  bile  acid  plus  sugar  by  mouth  causes  a 
delayed  bile  acid  excretion  with  fairly  low  volume  of  bile.  The 
extreme  cholagogue  action  of  bile  acids  may  be  completely 


386 


Metabolism  of  Bile  Acids.   '  III 


inhibited  by  simultaneous  administration  of  a  sugar  solution  by 
stomach.  Sugar  solutions  alone  will  cause  the  excretion  of  a  very 
concentrated  bile  but  will  not  modify  the  bile  acid  curve.  This 

TABLE  XXII. 

The  Effect  of  Taurine  and  Cholic  Acid  Separately  and  Combined. 
Dog  17-151.*    Simple  Bile  Fistula. 


Date. 

I  Volume. 

Amino 
nitrogen. 

Taurocholic 
acid  in  6 
hours  . 

<£> 

£ 

if 

MJ3 
fc 

I 

Urinary  N. 

Remarks. 

Inl 
cc.  of 
bile. 

In  6 

hours. 

1918 

cc. 

mg. 

mg. 

mg. 

mg. 

Ibs. 

gm. 

Average  11  days. 

15 

8.0* 

294 

3.3 

Hb.  138  per  cent. 

R.  B.C.  7,670  ,000. 

Mar.  30  

34 

0.277 

9.42 

346 

26.6 

36.75 

2.52 

Before  collection: 

4  gm.  of  cholic 

acid  in  capsule. 

"      31  

29 

0.133 

3.86 

142 

19.4 

36.60 

3.25 

Apr.     1  

18 

0.312 

5.61 

206 

23.5 

36.50 

2.41 

Before  collection: 

0.75  gm.  of  tau- 

rine  in  30  cc.  of 

salt  solution  in- 

travenously. 

2  

28 

0.353 

9.89 

366 

17.5 

36.25 

2.63 

3  

45 

0.797 

35.85 

1,316 

17.3 

35.80 

2.63 

Before  collection: 

3   gm.    of   mix- 

• 

ture     f     cholic 

acid,  |  taurine 

in  capsule. 

"        4  

19 

0.467 

8.88 

326 

18.2 

35.75 

2.30 

5  

47 

1.141 

53.60 

1,967 

20.9 

35.50 

2.58 

Before  collection: 

, 

4   gm.    of   tau- 

rocholic  acid  in 

capsule. 

"        6 

15 

0.402 

6.03 

220 

35.00 

2.52 

Hb.  125  per  cent. 

R.B.C.  6,980,000. 

*  This  dog  was  kept  on  a  diet  of  75  gm.  of  cane  sugar  and  100  gm.  of 
glucose  during  this  entire  experiment,  as  well  as  during  the  fore-period  of 
11  days. 

experiment  gives  the  highest  concentration  of  bile  acids  per  cc. 
of  fistula  bile  (86  mg.  per  cc.).  The  sugar  seems  responsible  for  a 
definite  delay  in  output  of  bile  acids;  but  the  output  is  enormous 
in  spite  of  the  low  volume  and  slight  delay  in  elimination. 


Foster,  Hooper,  and  Whipple 


387 


Tables  XXII  and  XXIII  are  companion  experiments  and  show 
great  uniformity  of  reaction.  From  these  two  and  many  other 
experiments  it  is  established  that  taurine  by  mouth  or  intraven- 

TABLE  XXIII. 

The  Effect  of  Taurine  and  Cholic  Acid  Separately  and  Combined. 
Dog  18-23*    Simple  Bile  Fistula. 


Date. 

Volume. 

Amino 
nitrogen. 

Taurocholic 
acid  in  6 
hours. 

Pigments  in  6 
hours. 

1 
I 

Urinary  N. 

Remarks  . 

Inl 
cc.  of 
bile. 

In  6 

hours. 

1918 

Average  12  days. 
Mar.  30  

cc. 
24 

45 

34 
40 

30 
55 

29 
56 

37 

mg. 

0.551 

0.183 
0.142 

0.240 
0.698 

0.354 
0.867 

0.171 

mg. 
8.7* 

24.75 

6.22 
5.68 

7.20 
38.4 

10.25 
48.53 

6.33 

mg. 

319 
909 

228 
208 

264 
1,300 

376 
1,780 

232 

mg. 

18.0 

24.6 
18.8 

14.6 
10.5 

11.1 
22.4 

14.8 

Ibs. 

27.7 

27.4 
27.1 

27.0 
26.6 

26.5 
26.4 

25.8 

gm. 

3.10 
3.27 

3.25 
2.57 

2.74 
2.55 

2.69 

2.58 

3.30 

Hb.  120  per  cent. 
R.  B.C.  7,895,000. 
Before  collection: 
3  gm.  of  cholic 
acid  in  capsule. 

Before  collection: 
0.75  gm.  of  tau- 
rine in  25  cc.  of 
salt  solution  in- 
travenously. 

Before  collection: 
2.6  gm.  of  mix- 
ture    I     cholic 
acid,   1  taurine 
in  capsule. 

Before  collection: 
3    gm.    of    tau- 
rocholic  acid  in 
capsule. 
Hb.  120  per  cent. 
R.  B.C.  7,300,000. 

"      31  

Apr.     1  

2  
"       3  

4  
"       5  

"        6      

*  This  dog  was  kept  on  a  diet  of  75  gm.  of  cane  sugar  and  75  gm.  of 
glucose  during  this  entire  experiment,  as  well  as  during  the  fore-period 
of  12  days. 

ously  does  not  in  any  way  influence  the  output  of  bile  acids.     This 
is  true  during  fasting  periods  as  well  as  for  full  diets. 

Taurocholic  acid  by  mouth  is  known  to  be  a  cholagogue  and  this 


388  Metabolism  of  Bile  Acids.     Ill 

action  is  well  shown  in  our  experiments.  About  40  to  50  per  cent 
of  the  taurocholic  acid  is  excreted  in  the  bile  during  the  6  hour 
collection  period.  There  may  have  been  slight  delay  in  excretion 
owing  to  the  use  of  capsules  rather  than  solutions. 

Taurine  plus  cholic  acid  fed  by  mouth  exerts  the  same  influence 
on  the  bile  fistula  dog  as  does  the  pure  taurocholic  acid.  In  other 
words  this  synthesis  can  take  place  in  the  body  with  great  ease  and 
rapidity.  The  cholagogue  action  and  increased  bile  acid  output 
are  identical,  whether  the  mixture  of  taurine  plus  cholic  acid  is 
given  or  the  pure  taurocholic  acid. 

Cholic  acid  alone  has  less  than  usual  influence  upon  the  bile  acid 
output  if  given  after  a  long  fasting  period.  There  may  be  a  little 
increase  in  bile  acid  excretion  or  there  may  be  a  decided  increase. 
The  reaction  seems  to  depend  upon  the  amount  of  taurine  present 
in  the  body  which  is  available  to  combine  with  the  cholic  acid 
radical.  Note  additional  data  in  Table  XXIV. 

Table  XXIV  furnishes  more  interesting  data  concerning  the 
feeding  of  cholic  acid  and  its  influence  upon  the  bile  acid  excretion. 
A  small  dose  (2.0  gm.)  of  cholic  acid  acts  as  a  cholagogue  but  does 
not  increase  the  output  of  bile  acid  after  an  11  day  fasting  period. 
A  larger  dose  (4.0  gm.)  of  cholic  acid  after  9  days  fasting  produces  a 
cholagogue  action  and  a  definite  increase  in  the  taurocholic  acid  of 
the  bile — an  increase  of  about  100  per  cent.  The  next  day  the 
same  dose  repeated  gives  the  same  cholagogue  action,  but  little  if 
any  increase  in  the  bile  acid  output.  We  may  assume  that  the  first 
dose  of  cholic  acid  combined  with  all  the  available  taurine  in  the 
body  and  formed  taurocholic  acid  to  be  eliminated  in  the  bile. 
The  second  large  dose  given  the  next  day  found  no  taurine  to  com- 
plement the  cholic  acid  and  no  taurocholic  acid  resulted.  We  see 
therefore  that  either  taurine  or  cholic  acid  can  act  as  limiting 
factors  in  the  over-production  of  taurocholic  acid.  This  is  par- 
ticularly true  of  cholic  acid,  which  is  probably  the  normal  deter- 
mining factor.  Cholic  acid  given  by  mouth  during  periods  of  full 
diet  is  usually  associated  with  a  strong  cholagogue  action  and  a 
great  increase  in  output  of  bile  acids  (Table  XXIV — Dog  17-34). 
This  indicates  an  abundant  exogenous  source  of  taurine  in  the 
common  mixed  diet. 

Table  XXV  shows  the  curve  which  results  from  the  feeding  of 
sodium  taurocholate.  Its  reaction  seems  to  be  identical  with  that. 


Foster,  Hooper,  and  Whipple 


389 


TABLE  XXIV. 
Cholic  Acid  Feeding. 


Amino 

O  CO 

nitrogen. 

Dog 

. 

J3-2     • 

"R                      \r 

No! 

JJate. 

a 

Inl 
cc.of 
bile. 

In  6 
hours 

jil 

f 

1918 

cc. 

mg. 

mg. 

mg. 

Ibs. 

15-22 

Average. 

16 

10.0 

367 

Average  3  days  after  11  days 

fasting. 

May  4 

35 

0.231 

8.09 

297 

28.0 

2.0   gm.    of    cholic   acid   in 

capsule  at  beginning. 

June  3 

16 

0.510 

8.16 

299 

29.5 

After  8  days  fasting. 

"     4 

38 

0.535 

20.33 

746 

29.0 

4.0   gm.   of   cholic   acid    in 

emulsion  at  beginning. 

"     5 

35 

0.250 

8.75 

321 

28.4 

4.0   gm.   of   cholic  '  acid   in 

emulsion  at  beginning. 

17-34 

Oct.    9 

24 

0.537 

12.90 

473 

31.5 

Mixed  diet. 

"     10 

114 

0.579 

66.00 

2,420 

31.5 

4.0   gm.   of   cholic   acid   in 

15   cc.   of  alcohol  at  be- 

ginning. 

"     11 

128 

0.478 

61.2 

2,250 

31.5 

4.0  gm.    of   cholic   acid   in 

15  cc.  of  alcohol  at  begin- 

ning. 

TABLE  XXV. 


Bile  Salt  Feeding. 
Dog  15-22.    Simple  Bile  Fistula. 


Amino 

O  CO 

a 

TT-,,_ 

nitrogen. 

Hour. 

1 

In  1  cc. 

In 

ill 

3  CP   O 

o>  ^ 

Remarks. 

> 

of  bile. 

2  hours. 

d  ^ 

£ 

cc. 

mg. 

mg. 

mg. 

mg. 

September  4,  1917. 

1-2 

18 

0.800 

14.14 

529 

6.8 

End  2nd  hour  10  gm.  of  crude 

sodium  taurocholate  given  by 

stomach. 

3-4 

58 

1.711 

99.26 

3,640 

18.4 

5-6 

45 

1.597 

71.85 

2,635 

11.7 

7-8 

22 

0.642 

14.12 

518 

8.4 

Weight  35.0  Ibs. 

390  Metabolism  of  Bile  Acids.     Ill 

observed  after  taurocholic  acid  feeding.  The  cholagogue  effect 
is  pronounced  and  about  50  per  cent  of  the  taurocholate  is  re- 
covered in  the  first  4  hours  of  collection.  The  crude  taurocholate 
was  not  carefully  prepared  or  analyzed,  so  that  the  actual  per- 
centage output  cannot  be  estimated.  The  reaction  subsides 
rapidly  and  falls  almost  to  normal  within  6  hours. 

DISCUSSION. 

Von  Bergman  (1)  fed  sodium  cholate  to  dogs  and  found  a  decided 
increase  in  the  sulfur  content  when  only  1.0  gm.  was  given.  But 
his  dog  weighed  only  4.5  kilograms.  A  dose  of  2.0  gm.  caused  an 
even  greater  increase,  but  did  not  double  it.  He  also  fed  a  dog  of 
8.5  kilograms  sodium  cholate  (2.0  gm.)  every  day  for  3  days.  The 
first  day  there  was  a  sufficient  amount  of  sulfur  excreted  to  account 
for  all  the  cholate  being  changed  into  taurocholic  acid.  But  each 
successive  day  the  sulfur  excreted  was  less.  The  third  day  it  was 
not  back  to  normal  although  it  was  much  lower.  He  explains  this 
by  saying  that  the  body  could  not  furnish  sufficient  taurine  to 
unite  with  the  abnormally  high  amount  of  sodium  cholate  given. 
Our  experiments  show  that  a  minimal  amount  of  taurine  is  avail- 
able after  a  long  fasting  period  (Table  XXIV).  Our  mixed  diet 
was  probably  richer  in  sulfur  than  von  Bergman's  diet  of  200  gm. 
of  meat,  150  gm.  of  rice,  and  30  gm.  of  casein.  Note  the  high  out- 
put with  cholic  acid  feeding  and  a  liberal  mixed  diet  (Table  XXIV). 

Goodman  (2)  gave  0.6  gm.  of  cholic  acid  to  a  dog  on  one  day  and 
found  a  decided  increase  in  the  amount  of  bile  and  the  cholic  acid 
excreted.  This  experiment  was  done  once  on  a  dog  of  4.5  kilo- 
grams weight  on  a  diet  of  dog  biscuit. 

The  cholic  acid  appears  to  be  the  important  determining  factor 
in  the  output  of  taurocholic  acid,  and  it  is  of  some  importance 
to  learn  the  source  of  cholic  acid  in  the  body,  its  true  metabolic 
history,  and  its  usefulness  and  ultimate  fate  in  the  body.  The 
solution  of  these  and  other  questions  relating  to  cholic  acid  is  not 
easy  but  will  repay  further  investigation. 

Feeding  cholic  acid  causes  a  minimal  reaction  after  long  periods 
of  fasting.  Repeated  doses  of  cholic  acid  during  fasting  result 
in  complete  failure  of  subsequent  doses  to  call  out  an  increase  of 
bile  acid  excretion.  This  may  be  interpreted  to  mean  that  under 


Foster,  Hooper,  and  Whipple  391 

these  conditions  of  fasting  and  repeated  ingestion  of  cholic  acid 
the  available  taurine  is  reduced  close  to  zero.  The  cholic  acid  is 
unable  to  combine  with  taurine  in  the  usual  way  and  is  eliminated 
in  some  other  form,  perhaps  in  the  bile  or  elsewhere. 

Feeding  cholic  acid  without  taurine  in  a  liberal  mixed  diet  will 
give  a  maximal  cholagogue  action  and  output  of  bile  acids.  Pre- 
sumably under  these  conditions  there  is  ample  taurine  to  combine 
with  the  cholic  acid.  The  result  is  a  large  increase  in  taurocholic 
acid  in  the  bile.  Probably  under  normal  conditions  of  diet  and 
health  there  is  always  available  an  excess  of  taurine  so  that  the 
normal  determining  factor  is  the  cholic  acid  radical.  Depending 
upon  the  available  supply  of  cholic  acid  there  is  a  high  or  low  out- 
put of  bile  acids  in  fistula  bile.  It  is  evident  that  certain  foods 
favor  a  high  bile  acid  output,  and  presumably  furnish  considerable 
amounts  of  cholic  acid  in  their  metabolic  history.  Much  more 
data  on  this  point  will  be  furnished  in  other  publications. 

SUMMARY. 

When  moderate  amounts  of  bile  are  given  by  mouth  (less  than 
1.8  gm.  of  taurocholic  acid)  about  90  per  cent  of  the  contained 
taurocholic  acid  is  excreted  in  the  first  4  hours. 

Larger  amounts  of  concentrated  bile  (8.0  to  11.0  gm.  of  tauro- 
cholic acid)  may  prolong  the  cholagogue  action  for  many  hours  or 
even  days. 

A  large  amount  of  concentrated  bile  given  with  sugar  causes  a 
very  high  concentration  of  bile  acids  in  the  bile  excreted  (7  to  9 
per  cent).  There  may  even  be  an  absence  of  cholagogue  action. 
This  may  represent  the  maximum  effort  of  the  liver  cell  to  con- 
centrate bile  acids  in  bile. 

Taurocholic  acid  and  sodium  taurocholate  given  by  mouth  have 
the  familiar  cholagogue  action  and  a  large  amount  will  appear  in 
the  bile  fistula  bile  (40  to  80  per  cent)  within  4  to  6  hours,  depend- 
ing upon  the  dose  given. 

Taurine  intravenously  has  no  effect  on  the  excretion  of  bile 
acids. 

Taurine  plus  cholic  acid  by  mouth  causes  a  marked  increase  in 
bile  secretion  and  bile  acid  output — as  much  as  does  taurocholic 
acid  itself. 


392  Metabolism  of  Bile  Acids.     Ill 

Cholic  acid  by  mouth  usually  causes  a  distinct  cholagogue  effect. 
Cholic  acid  fed  during  long  periods  of  fasting  gives  a  minimal 
output  of  bile  acids,  but  fed  during  full  diet  periods  gives  a  maximal 
output  of  bile  acids.  This  reaction  probably  depends  upon  the 
available  supply  of  taurine,  which  is  much  reduced  after  fasting, 
but  is  abundantly  available  during  full  diet  periods. 

We  are  indebted  to  Dr.  C.  L.  A.  Schmidt  for  a  large  amount  of 
taurine  which  was  prepared  in  his  laboratory. 

BIBLIOGEAPHY. 

1.  von  Bergman,  G.,  Beitr.  chem.  Physiol.  u.  Path.,  1904,  iv,  192. 

2.  Goodman,  E.  H.,  Beitr.  chem.  Physiol.  u.  Path.,  1907,  ix,  91. 

3.  Hooper,  C.  W.,  Am.  J.  Physiol.,  1917,  xlii,  280. 

4.  Hooper,  C.  W.,  and  Whipple,  G.  H.,  Am.  J.  Physiol.,  1917,  xlii,  264. 

5.  Rutherford,  W.,   and  Vignal,  M.,  J.  Anal,  and  Physiol.,  1876,  x,  253; 

ibid.,  1877,  xi,  61,  623. 

6.  Schiff,  M.,  Arch.  ges.  Physiol.,  1870,  iii,  598. 

7.  Socoloff,  N.,  Arch.  ges.  Physiol.,  1875,  xi,  166. 

8.  Stadelmann,  E.,  Z.  physiol.  Chem.,  1897,  xxxiv,  1. 

9.  Whipple,  G.  H.,  and  Hooper,  C.  W.,  Am.  J.  Physiol.,  1916,  xl,  349. 


THE  WAVERLY  P 

0ALTIMORH,  U.  «.  A. 


THE  METABOLISM  OF  BILE  ACIDS 
IV.    ENDOGENOUS  AND  EXOGENOUS  FACTORS 


BY 

M.  G.  FOSTER,  C.  W.  HOOPER,  AND  G.  H.  WHIFFLE 


(FROM  THE  GEORGE  WILLIAMS  HOOPER  FOUNDATION  FOR  MEDICAL 

RESEARCH,  UNIVERSITY  OF  CALIFORNIA  MEDICAL  SCHOOL, 

SAN  FRANCISCO) 


REPRINTED  FROM 

THE  JOURNAL  OF  BIOLOGICAL  CHEMISTRY 
VOL.  XXXVIII,  No.  2,  JUNE,  1919 


Reprinted  from  THE  JOURNAL  OF  BIOLOGICAL  CHEMISTRY,  Vol.  XXXVIII,  No.  2,  1919. 


THE  METABOLISM  OF  BILE  ACIDS. 
IV.     ENDOGENOUS  AND  EXOGENOUS  FACTORS. 

BY  M.  G.  FOSTER,  C.  W.  HOOPER,  AND  G.  H.  WHIPPLE. 

(From  the  George  Williams  Hooper  Foundation  for  Medical  Research, 
University  of  California  Medical  School,  San  Francisco.) 

(Received  for  publication,  April  28,  1919.) 

This  paper  gives  the  results  of  experiments  to  show  the  excre- 
tion of  bile  acids  during  periods  of  fasting,  of  sugar  feeding,  and 
of  standard  diets.  The  total  daily  urinary  nitrogen  excretion 
is  given  in  some  experiments  to  show  that  the  output  of  bile  acids 
runs  an  interesting  parallel  to  the  body's'  endogenous  nitrogen 
metabolism.  Limited  diets  may  profoundly  influence  the  curve 
of  bile  acid  excretion,  and  the  nitrogenous  portion  of  the  diet  is 
most  important.  It  is  obvious  that  certain  meat  proteins  added 
to  the  diet  profoundly  modify  the  excretion  of  bile  acids.  So 
there  seems  to  be  an  interesting  relationship  between  the  metab- 
olism of  meat  proteins  from  the  food  and  of  bile  acids  excreted 
in  the  bile.  It  is  at  least  possible  that  a  similar  relationship 
may  hold  for  the  tissue  proteins  of  the  body  and  the  bile  acid 
excretion.  Much  more  experimental  data  must  be  submitted 
but  the  experiments  outlined  below  make  certain  fundamental 
points  quite  clear. 

Bidder  and  Schmidt  (1)  followed  the  solid  constituents  of  the  bile  and 
concluded  that  there  was  an  increase  on  a  meat  diet.  Spiro  (3)  followed 
the  sulfur  in  the  food  and  in  the  bile  and  found  that  increasing  amounts 
of  meat  increased  the  sulfur  excreted,  but  not  proportionately.  He  also 
stated  that  there  was  a  continuous  sulfur  excretion  during  fasting,  but 
at  a  much  lower  level  than  when  fed.  Also,  feeding  carbohydrates  de- 
creases the  amount  of  sulfur  excreted.  These  experiments  are  in  accord 
with  our  results. 

Kunkel  (2)  followed  the  sulfur  partition  in  a  single  day  and  found  that 
the  sulfur  increased  in  the  bile. on  starvation  at  one  time,  and  not  at 
another.  Bread  and  milk  decreased  the  sulfur  while  various  amounts  of 
meat  did  not  have  any  effect  at  all.  Twice  that  amount  of  blood  by 
stomach  decreased  the  bile  sulfur.  The  dog  lived  only  about  a  month 
after  the  experiment,  so  that  very  little  importance  can  be  attached  to 
these  experiments. 

orvo 

393 

THE  JOURNAL   OP   BIOLOGICAL,  CHEMISTRY,    VOL.   XXXVIII,    NO.  2 


394 


Metabolism  of  Bile  Acids.     IV 


EXPERIMENTAL. 

These  dogs  were  kept  on  the  usual  routine  already  described 
for  the  previous  articles  of  this  series.  The  experiments  given 
in  Tables  XXXI  to  XXXIV  show  the  urinary  nitrogen  excretion 

TABLE  XXXI. 

Sugar  Feeding. 
Dog  17-151.    Simple  Bile  Fistula. 


Amino 

CO 
CJ 

I" 

a 

nitrogen. 

O.S 

1  * 

&B 

•°        in 

•x  3 

Date. 

I 

In  1 

O 

8^  5 

•aS 

$2 

4* 

.C 

KemarKs  . 

3 

2 

cc.of 
bile. 

In  6 
hours. 

I'iJi 

,2  o 

is 

i 

1918 

cc. 

mg. 

mg. 

mg. 

mg. 

gm. 

Ibs. 

Mar.  18 

13 

0.815 

10.59 

389 

38.4 

43.0 

75  gm.  of  cane  sugar,  50 

gm.  of   glucose  given 

daily  by  stomach  tube. 

"     19 

16 

0.618 

9.88 

363 

69.5 

4.76 

40.7 

Hb.  125  per  cent. 

R.  B.  C.  6,840,000. 

"     20 

11 

0.972 

10.69 

392 

42.0 

3.05 

40.8 

"     21 

10 

0.802 

8.02 

294 

20.9 

3.16 

39.7 

No  bile  exclusion  in  this 

experiment. 

"     22 

3.89 

38.7 

"     23 

13 

0.780 

10.14 

372 

36.6 

2.71 

39.0 

75  gm.  of  cane  sugar,  100 

gm.  of  glucose  given 

•  *- 

daily  by  stomach  tube. 

"     24 

12 

0.482 

5.78 

212 

37.6 

3.33 

38.4 

"     25 

11 

0.556 

6.11 

224 

21.0 

3.92 

38.5 

"     26 

15 

0.379 

5.68 

209 

26.1 

3.42 

38.0 

"     27 

30 

0.236 

7.08 

260 

14.6 

3.41 

37.9 

"     28 

20 

0.279 

5.58 

205 

29.2 

3.17 

37.25 

Hb.  138  per  cent. 

R.  B.  C.  7,670,000. 

"     29 

19 

0.472 

8.98 

329 

30.8 

3.11 

37.0 

Average  

8.05 

295 

3.45 

86  mg.  of  bile  acid  per  1.0  gm. 

of  urinary  nitrogen. 

per  24  hour  periods.  In  these  experiments  the  dogs  were  kept 
at  all  times  in 'standard  metabolism  cages  arranged  for  complete 
collection  of  the  urine  with  elimination  of  the  feces.  Diarrhea 
was  never  present  and  no  fecal  nitrogen  is  included  in  these 
figures.  The  night  bile  of  course  was  included  in  urine  collections, 
but  the  nitrogen  concerned  is  constant  and  rarely  exceeds  0.2  gm. 


Foster,  Hooper,  and  Whipple 


395 


per  24  hours.  The  dogs  were  catheterized  at  the  same  hour  each 
day,  weighed,  and  given  the  sugar  solutions  or  water  by  stomach 
tube.  The  cage  was  then  washed  out  and  the  washings  added 
to  the  cage  urine,  bladder  urine,  and  bladder  washings,  which 
were  made  up  to  a  unit  volume.  Duplicate  specimens  were 
analyzed  by  the  Kjeldahl  method  and  the  total  nitrogen  calcu- 

TABLE  XXXII. 

Fasting. 
Dog  17-151.    Simple  Bile  Fistula. 


Amino 

on 

c 

nitrogen. 

.2  eo 

£  £ 

£  gj 

. 

%*» 

II 

£3  ' 

• 

Date. 

1 

Inl 
cc.  of 
bile. 

In  6 
hours. 

o     2 

H  °3' 

£™ 

I 

Remarks. 

1918 

cc. 

mg. 

mg. 

mg. 

mg. 

gm. 

76s. 

April  23 

38 

0.513 

19.50 

716 

13.2 

40.7 

No  bile  exclusion  in  this 

experiment. 

"     24 

18 

0.510 

9.18 

337 

41.3 

2.24 

38.6 

Hb.  132  per  cent. 

R.  B.  C.  6,800,000. 

"     25 

15 

0.704 

10.56 

388 

33.9 

4.14 

37.4 

"     26 

15 

0.508 

7.62 

279 

24.9 

3.97 

36.6 

"     27 

11 

0.615 

6.76 

248 

15.2 

4.20 

36.0 

Set  up  3  hours. 

"     28 

27 

16.0 

4.62 

34.5 

"     «   3      « 

"     29 

25 

0.543 

13.68 

502 

13.1 

4.20 

35.0 

"     30 

15 

0.641 

9.61 

352 

12.0 

3.83 

34.5 

May      1 

18 

0.711 

12.78 

468 

12.5 

3.55 

34.13 

"       2 

16 

0.899 

14.39 

528 

9.5 

3.78 

33.56 

"       3 

15 

0.676 

10.15 

373 

11.8 

3.97 

33.13 

Hb.  130  per  cent. 

R.  B.  C.  7,235,000. 

Average  

11.42 

419 

3.85 

109  mg.  of  bile  acid  per  1.0  gm- 

of  urinary  nitrogen. 

lated.  One  of  the  dogs  (15-22,  Table  XXXIV)  has  an  obstruction 
in  his  urethra  which  makes  catherization  impossible.  The 
regular  24  hour  collections  were  made  as  usual,  and  the  average 
of  several  days  will  correct  for  the  daily  variations  which  are  in 
part  due  to  variable  amounts  of  bladder  urine  retained  on  dif- 
ferent days. 

Tables  XXXI  and  XXXII  are  to  be  compared,  as  these  experi- 
ments were  performed  upon  the  same  healthy,  vigorous  dog  under 


396  Metabolism  of  Bile  Acids.     IV 

identical  experimental  conditions.  The  amount  of  sugar  given 
in  the  first  experiment  was  not  large  for  a  dog  of  this  size,  and 
was  increased  from  125  gm.  per  day  to  175  gm.  during  the  last 
hah0  of  the  experiment.  It  may  be  merely  a  coincidence,  but 
during  the  second  period  of  higher  sugar  intake  the  bile  acid 
output  fell  somewhat.  This  dog  averaged  3.45  gm.  of  urinary 
nitrogen  per  day  and  295  mg.  of  taurocholic  acid  per  6  hours. 
The  6  hour  amount  of  taurocholic  acid  per  gm.  of  daily  nitrogen 
is  therefore  86  mg. 

When  this  dog  is  put  on  fasting  after  a  suitable  resting  period 
with  liberal  mixed  diet  we  note  a  higher  urinary  nitrogen  output, 
3.85  gm.  per  day- and  419  mg.  of  taurocholic  acid  per  6  hours. 
The  amount  per  gm.  of  24  hour  nitrogen  is  therefore  109  mg. 
taurocholic  acid  per  6  hour  period.  As  the  endogenous  urinary 
nitrogen  excretion  rises  with  fasting,  as  compared  with  sugar 
feeding,  we  note  a  parallel  or  slightly  greater  rise  in  the  output 
of  taurocholic  acid. 

We  wish  to  point  out  an  interesting  fact  in  Tables  XXXII 
and  XXXIV.  The  first  day  in  each  table  gives  a  remarkably 
high  taurocholic  acid  figure,  and  this  may  not  be  clear  until  one 
remembers  that  a  full  mixed  diet  preceded  this  fasting  period. 
The  first  day's  bile  acid  output  results  from  the  mixed  diet  of 
the  preceding  day,  but  it  is  significant  that  after  the  first  24  hours 
the  base  line  is  reached  and  maintained.  This  is  somewhat  dif- 
ferent from  the  basal  nitrogen  excretion,  which  does  not  reach 
its  lowest  level  on  fasting  until  the  third  or  fourth  day. 

Tables  XXXIII  and  XXXIV  are  like  the  preceding  two  ex- 
periments and  correspond  in  almost  every  detail.  These  two 
experiments  were  done  on  two  different  dogs  of  approximately 
the  same  weight.  Both  were  in  excellent  condition.  The  fast- 
ing dog  of  course  shows  a  higher  output  of  urinary  nitrogen  and 
also  of  taurocholic  acid  per  6  hour  period.  The  fasting  dog  shows 
a  urinary  nitrogen  of  3.76  gm.  per  24  hours  and  taurocholic  acid 
407  mg.  per  6  hours.  The  sugar  fed  dog  presents  a  urinary  ni- 
trogen of  3.23  gm.  per  24  hours  and  taurocholic  acid  318  mg.  per 
6  hours.  The  amount  of  6  hour  taurocholic  acid  per  gm.  of  24 
hour  nitrogen  is  almost  identical,  108  mg.  in  fasting  and  98  mg. 
with  sugar. 

It  seems  sufficiently  clear  that  there  is  a  close  relationship 


Foster,  Hooper,  and  Whipple 


397 


•between  the  endogenous  nitrogen  metabolism  and  the  excre- 
tion of  taurocholic  acid  in  bile  fistula  bile.  During  fasting  periods 
we  may  assume  that  more  body  protein  is  broken  down  and  more 
taurocholic  acid  results  from  this  process.  We  may  assume  that 

TABLE  XXXIII. 

Sugar  Feeding. 
Dog  18-23.    Simple  Bile  Fistula. 


Amino 

I* 

a 

nitrogen. 

J.s  . 

1  1 

*  a 

Date. 

• 

II 

bo 

^ 

Remarks. 

Tn  1 

O««o 

^ 

1 

in  i 
cc.of 
bile. 

In  6 
hours 

JH 

-  s 

s 

1* 

I 

1918 

cc. 

mg. 

mg. 

mg. 

mg. 

gm. 

Ibs. 

Mar.  18 

52 

0.204 

10.60 

389 

26.2 

33.3 

75  gm.  of  cane  sugar,  50 

gm.  of  glucose  given 

* 

daily      by      stomach 

tube. 

"     19 

27 

0.329 

8.88 

326 

17.4 

4.51 

31.75 

Hb.  118  per  cent. 

R.  B.  C.  6,130,000. 

"     20 

31 

0.324 

10.04 

368 

15.6 

3.72 

31.06 

No  bile  exclusion  in  this 

experiment. 

"     21 

17 

0.507 

8.62 

316 

10.1 

3.08 

30.75 

"     22 

20 

0.414 

8.28 

304 

13.9 

3.88 

29.19 

"     23 

22 

0.288 

6.33 

232 

10.7 

3.22 

29.75 

75  gm.  of  cane  sugar,  75 

gm.  of  glucose  given 

daily      by      stomach 

tube. 

"     24 

17 

0.512 

8.70 

319 

14.2 

2.04 

29.56 

"     25 

23 

0.394 

9.06 

333 

22.0 

3.64 

29.44 

"     26 

26 

0.325 

8.45 

310 

11.3 

3.13 

28.0 

"     27 

33 

0.194 

6.40 

235 

12.6 

3.19 

28.56 

"     28 

20 

0.363 

7.26 

266 

10.4 

2.74 

27.38 

Hb.  120  per  cent. 

R.  B.  C.  7,895,000. 

"     29 

34 

0.332 

11.30 

415 

14.2 

2.38 

27.06 

Average 

8.66 

318 

3.23 

98  mg.  of  bile  acid  per  1.0 

gm.  of  urinary  nitrogen. 

sugar  feeding  enables  the  body  to  conserve  its  protein  at  the  source, 
or  enables  the  body  to  conserve  its  protein  end-products  and  re- 
construct these  into  body  cells.  When  less  bile  acids  are  excreted 
during  sugar  periods  we  may  wish  to  assume  some  such  con- 
servation of  bile  acid  or  its  parent  substance  for  other  uses  in 


398 


Metabolism  of  Bile  Acids.     IV 


body  metabolism.  Refer  also  to  Table  XL,  where  there  is  even- 
more  conservation  of  bile  acids  or  substances  from  which  they  are 
derived. 

Fasting  does  not  decrease  the  bile  acid  output  to  a  remarkably 
low  level.  Sugar  feeding  causes  a  drop  of  bile  acid  excretion 
below  the  fasting  level.  These  two  points  emphasize  the  fact 

TABLE  XXXIV. 

Fasting. 
Dog  15-22.    Simple  Bile  Fistula. 


Amino 

9 

g 

nitrogen. 

^3° 

11 

£  M 

o  g 

H    5 

Date. 

a 

JM 

bo 

-^ 

Remarks. 

"o 

Inl 
cc.  of 
bile. 

In  6 
hours  . 

Jll 

ft«o 
ra-2 

5M 

j 

1918 

cc. 

mg. 

mg. 

mg. 

mg. 

ffw. 

Ibs. 

April  23 

50 

0.668 

33.40* 

1,225* 

21.8 

34.7 

Dog   not   catheterized 

during  experiment. 

"     24 

20 

0.602 

12.04 

442 

20.9 

5.71 

33.0 

Hb.  150  per  cent. 

R.  B.  C.  6,390,000. 

"     25 

27 

0.470 

12.68 

465 

32.9 

3.75 

32.38 

No    bile     exclusion  in 

this  experiment. 

"     26 

18 

0.486 

8.77 

322 

21.9 

3.16 

31.5 

"     27 

28 

0.536 

15.00 

550 

4.4 

2.91 

31.4 

Set  up  3  hours. 

"     28 

27 

0.552 

14.90 

547 

10.3 

4.59 

30.56 

«           ((      Q             (( 

"     29 

22 

0.493 

10.85 

398 

4.2 

3.58 

30.20 

"     30 

20 

0.404 

8.09 

297 

22.6 

2.94 

29.63 

May      1 

17 

0.505 

8.58 

315 

26.6 

3.19 

29.20 

"       2 

12 

0.744 

8.93 

328 

16.3 

4.03 

28.70 

Average  

11.09 

407 

3.76 

108  mg.  of  bile  acid  per  1.0 

gm.  of  urinary  nitrogen. 

*  Not  included  in  average. 

that  there  is  an  important  endogenous  factor  in  the  bile  acid 
metabolism. 

Tables  XXXV  and  XXXVI  are  identical  in  practically  every  re- 
spect and  show  a  remarkable  parallelism  between  the  food  in- 
take nitrogen  and  the  output  of  taurocholic  acid.  It  will  be 
shown  later  that  this  reaction  depends  in  part  upon  the  type  of 
food  protein.  The  first  dog  (Table  XXXV)  was  in  perfect  con- 
dition during  the  entire  experiment,  maintained  a  constant  weight, 


Foster,  Hooper,  and  Whipple 


399 


TABLE  XXXV. 

Nitrogen  in  Food  and  Bile  Acid  Excretion. 
Dog  17-151.    Simple  Bile  Fistula. 


Amino 

5  . 

nitrogen. 

la 

<jj  M 

Date. 

1 

Inl 

In  6 

$8.8 

'&<o 

9 

Remarks. 

"o 

cc.  of 

hours. 

c«  S-^ 

JH    £5 

1 

bile. 

H 

ta 

£ 

1918 

cc. 

mg. 

mg. 

mg. 

mg. 

Us. 

Jan.    7 

43 

0.403 

17.32 

.636 

21.4 

38.0 

Diet  375  gm.  of  cracker  meal, 

90  gm.  of  beef  heart  contain- 

ing.100  calories  and  0.5  gm. 

of  nitrogen  per  kilo. 

"       8 

30 

0.575 

17.25 

633 

23.0 

38.0 

Hb.  135  per  cent. 

R.  B.  C.  7,576,000. 

"       9 

49 

0.246 

12.05 

442 

24.7 

38.0 

"     10 

32 

0.505 

16.16 

593 

10.6 

38.5 

"     11 

28 

0.311 

8.70 

319 

6.3 

38.5 

"     14 

20 

0.680 

13.60 

499 

20.8 

38.5 

"     15 

65 

0.260 

16.90 

620 

20.1 

39.0 

No  bile  exclusion  in  this  experi- 

ment. 

"     16 

28 

0.766 

21.44 

787 

18.5 

39.0 

"     17 

37 

0.538 

19.90 

730 

19.9 

39.3 

"     18 

17 

0.785 

13.35 

490 

16.0 

38.8 

Diarrhea. 

"     21 

16 

0.604 

9.66 

354 

18.9 

39.0 

"     22 

23 

0.591 

13.59 

499 

18.4 

39.0 

Average  

14.99 

550 

18.2 

Diet  0.5  gm.  of  nitrogen  per  kilo. 

Jan.  23 

67 

0.415 

27.80 

1,020 

23.6 

39.2 

696    gm.    of    beef    heart,    100 

calories  and  1.0  gm.  of  nitro- 

gen per  kilo. 

"     24 

50 

0.460 

27.00 

994 

14.8 

39.5 

"     25 

21 

0.500 

10.50 

386 

1.8 

39.0 

"     28 

37 

0.675 

24.97 

918 

3.9 

39.3 

"     29 

25 

1.016 

25.40 

934 

1.7 

39.0 

"     30 

32 

0.898 

28.73 

1,055 

2.8 

39.3 

"     31 

37 

0.733 

27.12 

998 

7.7 

39.0 

Feb.    1 

44 

0.729 

32.09 

1,177 

20.7 

39.0 

•  * 

"       4 

51 

0.645 

32.89 

1,206 

8.0 

39.0 

"       6 

18 

1.083 

19.49 

715 

27.6 

39.0 

"       7 

40 

0.871 

34.84 

1,279 

7.7 

38.8 

"       8 

35 

0.870 

30.45 

1,118 

10.4 

39.0 

Average 

i 

26.77 

982 

10.9 

Diet  1.0  gm.  of  nitrogen  per  kilo. 

400 


Metabolism  of  Bile  Acids.     IV 


TABLE  XXXVI. 

Nitrogen  in  Food  and  Bile  Acid  Excretion. 
Dog  18-23.    Simple  Bile  Fistula. 


Amino 

3 

nitrogen. 

a 

II 

Date. 

8 

-§'~  « 

Is 

_t>- 

Remarks. 

s 

3 

•3 

In  1  cc. 
of  bile. 

In  6 

hours. 

|l! 

5.50 

-S.2 

W 

1918 

cc. 

mg. 

mg. 

mg. 

mg. 

Ibs. 

Jan.    7 

42 

0.272 

11.02 

405 

40.9 

33.0 

Diet  325  gm.  of  cracker  meal, 

80  gm.  of  beef  heart  contain- 

ing 0.5  gm.  of  nitrogen  and 

100  calories  per  kilo. 

".      8 

62 

0.158 

9.79 

359 

27.0 

33.3 

"       9 

72 

0.073 

5.22 

191 

25.0 

33.0 

Hb.  115  per  cent. 

R.  B.  C.  7,024,000. 

"     10 

67 

0.206 

13.80 

506 

29.4 

33.0 

"     11 

69 

0.158 

10.93 

401 

17.6 

32.8 

No  bile  exclusion  in  this  ex- 

periment. 

"     14 

67 

0.143 

9.58 

453 

20.8 

33.0 

"     15 

76 

0.088 

6.68 

245 

26.4 

33.0 

"     16 

56 

0.211 

11.81 

433 

14.6 

33.5 

"     17 

74 

0.057 

4.21 

154 

18.1 

33.1 

"     18 

70 

0.184 

12.85 

472 

12.2 

33.3 

"     21 

75 

0.202 

15.13 

555 

18.3 

33.5 

"     22 

68 

0.226 

15.40 

566 

19.9 

33.5 

Average           .... 

10.53 

395 

22.5 

Diet  0.5  gm.  of  nitrogen  per  kilo. 

Jan.  23 

79 

0.3431 

27.1 

994 

15.2 

33.8 

602    gm.    of    beef    heart,    100 

calories  and  1.0  gm.  of  nitro- 

gen per  kilo. 

"     24 

77 

0.246 

18.93 

685 

18.8 

33.6 

"     25 

82 

0.217 

17.77 

552 

16.7 

33.0 

"     28 

43 

0.392 

16.85 

618 

11.0 

32.0 

350  gm.  not  eaten.     Diarrhea. 

"     29 

58 

0.416 

24.12 

885 

9.1 

31.8 

250    "       "        "                 " 

"     30 

55 

0.489 

26.89 

986 

22.0 

30.5 

110    "       "        " 

"     31 

70 

0.424 

29.68 

1,089 

20.7 

30.5 

on        «          ••             •  •                        n 

Feb.    1 

68 

0.380 

25.84 

948 

18.2 

30.3 

"       4 

80 

0.312 

24.96 

916 

18.1 

29.5 

Diarrhea. 

"       5 

60 

0.395 

23.70 

870 

14.6 

29.0 

"           Noon  meal  omitted. 

"       6 

48 

0.317 

15  21 

558 

8.4 

29.5 

"       7 

74 

0.213 

15  76 

578 

14.1 

29.0 

"       8 

47 

0.307 

14.42 

529 

11.5 

28.5 

Average  21.63 

794 

15.2 

Diet  1.0  gm.  of  nitrogen  per  kilo. 

Foster,  Hooper,  and  Whipple  401 

and  ate  all  food.  The  bile  pigment  figures  are  included  and  show 
remarkable  and  inexplicable  fluctuation,  particularly  with  the 
beef  heart  diet. 

The  taurocholic  acid  output  is  quite  uniform  each  day  with 
occasional  fluctuations.  The  average  daily  output  of  bile  acid 
per  6  hour  period  shows  a  very  remarkable  increase  when  the  dog 
was  suddenly  changed  from  the  diet  rich  in  carbohydrate  (0.5 
gin.  of  nitrogen  per  kilo)  to  the  beef  heart  diet  (1.0  gm.  of  nitrogen 
per  kilo).  The  increase  in  bile  acid  corresponds  to  the  increase 
in  food  nitrogen — that  is,  about  100  per  cent  increase.  The 
sharp  rise  in  taurocholic  acid  following  the  change  to  a  rich  pro- 
tein diet  (beef  heart)  is  well  shown  in  both  these  experiments. 
The  reaction  even  goes  above  the  average  figures  of  taurocholic 
acid  output  on  the  very  first  day  of  rich  protein  diet. 

The  second  dog  (Table  XXXVI)  shows  a  general  reaction  which 
is  in  every  respect  similar  to  the  preceding  experiment  but  for  some 
diarrhea  in  the  beef  heart  period.  Associated  with  this  was  some 
loss  of  weight  and  appetite.  These  abnormal  factors,  however, 
did  not  influence  the  uniform  reaction  to  the  change  in  food  pro- 
tein. Additional  data  which  confirm  these  experiments  will  be 
found  in  Paper  V  of  this  series. 

Tables  XXXVII  and  XXXVIII  add  some  interesting  data  to 
that  of  the  preceding  experiments.  In  both  these  experiments 
the  food  nitrogen  was  decreased  to  0.25  gm.  per  kilo  by  decreas- 
ing the  cracker  meal  and  replacing  the  beef  heart  with  fat.  The 
caloric  value  was  held  unchanged  at  100  calories  per  kilo.  One 
dog  (Table  XXXVII)  showed  practically  no  reaction  to  this 
change  in  diet  and  excreted  almost  the  same  amount  of  tauro- 
cholic acid  as  formerly  upon  a  diet  of  0.5  gm.  of  nitrogen  per  kilo. 
The  other  dog  (Table  XXXVIII)  showed  a  decided  drop  in  excre- 
tion of  taurocholic  acid  but  not  to  one-half  the  output  on  the 
diet  containing  0.5  gm.  of  nitrogen  per  kilo.  The  level  of  bile 
acid  output  on  this  cracker  meal-fat  diet  approaches  the  fasting 
excretion  level  of  bile  acid.  This  shows  at  once  that  there  is 
no  hard  and  fast  parallel  between  the  nitrogen  intake  and  bile 
acid  excretion,  but  it  is  clear  that  there  is  an  important  exogen- 
ous factor. 

The  mixed  diet  periods  show  a  level  of  bile  acid  excretion 
which  is  below  that  of  the  beef  heart  diet.  The  elements  in  the 


402 


Metabolism  of  Bile  Acids.     IV 


TABLE  XXXVII. 

Nitrogen  in  Food  and  Bile  Acid  Excretion. 
Dog  17-151.    Simple  Bile  Fistula. 


Amino 

CO 

.2   . 

nitrogen. 

a  c 

§g 

Date. 

• 

fP  a 

11 

Remarks. 

S 

In  1 

T!  3 

"CLCD 

43 

In  6 

-•s  § 

a 

1. 

cc.  of 
bile. 

hours. 

I8- 

§'" 

I 

1918 

cc. 

mg. 

mg. 

mg. 

mg. 

Ibs. 

Feb.  11 

32 

0.525 

16.80 

617 

38.8 

40.0 

262  gm.  of  cracker  meal,  10 

gm.  of  butter,  30  gm.  of 

lard,    105    gm.    of    cane 

sugar,  0.25  gm.  of  nitro- 

gen, and  100  calories  per 

kilo. 

"       12 

30 

0.536 

16.08 

590 

28.2 

39.5 

t 

"       13 

11 

0.602 

6.62 

243 

10.4 

39.8 

Hb.  125  per  cent. 

R.  B.  C.  6,240,000. 

"       14 

32 

0.425 

13.60 

499 

20.6 

40.0 

"       15 

24 

0.527 

12.66 

465 

20.9 

40.0 

"       18 

32 

0.385 

12.32 

452 

10.0 

40.0 

No  bile   exclusion   in   this 

experiment. 

"       19 

20 

0.493 

9.86 

362 

13.2 

40.0 

"       20 

45 

0.345 

15.52 

570 

14.9 

"       21 

23 

0.695 

15.98 

586 

5.2 

Average             

13.27 

487 

18.0 

Diet  0.25   gm.   of  nitrogen    per 

kilo. 

Mar.    5 

61 

0.534 

32.55 

1,196 

16.6 

42.5 

Mixed  diet. 

"         6 

48 

0.662 

.31.79 

1,167 

21.5 

41.8 

7 

80 

0.290 

23.19 

852 

36.9 

42.5 

Hb.  118  per  cent. 

R.  B.  C.  6,350,000. 

8 

32 

0.695 

22.25 

818 

10.1 

43.0 

"       11 

38 

0.286 

10.86 

399 

21.9 

41.5 

"       12 

30 

0.771 

23.14 

850 

3.9 

43.0 

: 

"       13 

28 

0.786 

22.00 

809 

11.3 

42.5 

"       14 

40 

0.616 

24.64 

906 

5.8 

43.5 

"       15 

22 

0.691 

15.20 

559 

12.6 

44.0 

Average  

22.85 

839 

15.6 

Mixed  diet. 

Foster,  Hooper,  and  Whipple 


403 


TABLE'  XXXVIII. 

Nitrogen  in  Food  and  Bile  Acid  Excretion. 
Dog  18-23.    Simple  Bile  Fistula. 


Amino 

CO 

-2  . 

nitrogen. 

Q 

§  £ 
S  3 

Date. 

o 

6 

Inl 

In  6 

'0.0 

I 

Remarks. 

1 

cc.  of 

bile. 

hours. 

|8J 

-.2 

fe 

1918 

cc. 

mg. 

mg. 

'mg. 

mg. 

Ibs. 

Feb.    11 

48 

0.231 

11.08 

407 

18.4 

30.3 

30  gm.  of  lard,  59  gm.  of 

cane    sugar,    198    gm.    of 

cracker  meal,   10  gm.  of 

butter,  0.25  gm.  of  nitro- 

gen, and  100  calories  per 

kilo. 

"       12 

43 

0.174 

7.47 

274 

16.1 

29.5 

"       13 

68 

0.100 

6.80 

253 

16.6 

29.5 

Hb.  115  per  cent. 

R.  B.  C.  7,928,000. 

"       14 

58 

0.027 

1.60 

58 

18.2 

29.3 

"       15 

63 

0.129 

8.10 

297 

18.4 

29.3 

No  bile  exclusion  in  this  ex- 

periment. 

"       18 

35 

0.114 

3.97 

145 

13.4 

29.0 

"       19 

64 

0.197 

12.64 

464 

11.1 

29.2 

"       20 

55 

0.172 

9.48 

348 

19.1 

"       21 

51 

0.085 

4.34 

159 

Average  

7.27 

267 

16.4 

Diet  0.25   gm.   of  nitrogen   per 

kilo. 

Mar.     5 

75 

0.241 

18.06 

664 

31.8 

32.0 

Mixed  diet. 

6 

64 

0.249 

15.93 

585 

36.3 

7 

85 

0.173 

14.73 

541 

37.0 

33.3 

Hb.  110  per  cent. 

R.  B.  C.  6,290,000. 

"         8 

81 

0.210 

17.04 

626 

37.5 

34.0 

"       11 

91 

0.200 

18.22 

670 

27.9 

32.8 

"       12 

69 

0.184 

12.72 

467 

29.0 

34.0 

"       13 

82 

0.324 

26.60 

978 

25.3 

35.0 

"       14 

60 

0.300 

18.01 

662 

27.6 

35.5 

"       15 

82 

0.307 

25.14 

923 

29.5 

34.8 

Average  

18.50 

679 

31.3 

Mixed  diet. 

404  Metabolism  of  Bile  Acids.     IV 

mixed  diet  are  of  course  variable.  This  diet  is  a  mixture  of 
kitchen  scraps — bones,  meat,  bread,  potato,  rice,  etc. 

Table  XXXIX  shows  a  third  dog  which  was  placed  upon  these 
same  diets  containing  0.5,  1.0,  and  0.25  gm.  of  nitrogen  per  kilo. 
This  dog  remained  in  perfect  condition  during  the  entire  experi- 
ment and  adds  confirmatory  data  to  the  other  experiments. 
This  bile  fistula  dog  has  been  under  observation  for  3  years,  and 
it  is  known  that  there  is  a  small  communication  between  the  com- 
mon duct  and  duodenum.  This  allows  a  small  amount  of  bile 
to  enter  the  duodenum  when  the  fistula  is  not  draining  freely, 
.for  example  at  night.  This  dog  shows  only  a  50  per  cent  rise 
in  bile  acid  output  when  the  diet  is  changed  from  0.5  to  1.0  gm. 
of  nitrogen  per  kilo.  Further  change  in  diet  from  1.0  to  0.25 
gm.  of  nitrogen  per  kilo  causes  a  fall  from  1,262  mg.  of  taurocholic 
acid  to  684  mg.  per  6  hour  period.  These  fluctuations  are  not 
proportional  to  the  nitrogen  content  of  the  diets,  but  it  is  obvious 
that  a  rise  in  the  food  nitrogen  intake  does  cause  a  rise  in  the 
taurocholic  acid  excretion  and  vice  versa. 

Table  XL  gives  the  data  on  two  experiments  which  show  the 
influence  of  a  preceding  period  of  fasting  upon  subsequent  excretion 
of  bile  acids  with  a  standard  diet  of  0.5  gm.  of  nitrogen  per  kilo. 
It  is  to  be  recalled  that  these  same  two  dogs  on  a  previous  occasion 
showed  an  output  of  bile  acids  on  this  same  diet  which  was  much 
higher  (550  and  875  mg.  of  taurocholic  acid  per  6  hour  period — 
Tables  XXXV  and  XXXIX) .  Compare  with  this  high  output  the 
low  excretion  in  Table  XL  on  the  same  diet  (376  and  419  mg.  of 
taurocholic  acid  per  6  hour  period).  The  only  factor  which  can 
explain  this  difference  is  the  preceding  fasting  period  of  10  and  11 
days.  This  low  level  of  excretion  is  actually  that  of  the  fasting 
period  or  even  lower.  It  is  evident  that  the  fasting  period  has 
caused  a  clianged  reaction  in  the  body  so  that  much  less  bile  acid 
is  permitted  to  escape  in  the  bile.  It  may  be  a  correct  assumption 
that  some  of  the  material  which  under  usual  diet  conditions  goes 
to  form  the  bile  acid  fraction  is  deviated  for  other  uses  in  the  body. 

This  reaction  must  be  kept  in  mind  whenever  any  diet  experi- 
ments are  planned  for  these  bile  fistula  dogs.  For  certain  experi- 
ments it  might  be  assumed  that  a  preliminary  period  of  fasting 
might  give  an  ideal  simple  base  line  from  which  to  estimate  the 
change  brought  about  by  a  given  diet.  But  the  reaction  may  be 


Foster,  Hooper,  and  Whipple 


405 


TABLE  XXXIX. 

Nitrogen  in  ^ood  and  Bile  Acid  Excretion. 
Dog  15-22.    Simple  Bile  Fistula. 


Amino 

CO 

1 

nitrogen. 

1.3 

Bo 

Date. 

1 

Inl 

In  6 

ill 

•22 

i 

Remarks. 

1 

cc.of 
bile. 

hours. 

i8* 

:§.S 

1 

1918 

cc. 

mg. 

mg. 

mg. 

mg. 

Ibs. 

Jan.     7 

13 

0.835 

10.85 

398 

10.0 

32.0 

325  gm.  of  cracker  meal,  75 

gm.    of   beef  heart.    100 

calories   and  0.5   gm.   of 

nitrogen  per  kilo. 

8 

20 

0.992 

19.84 

728 

3.5 

32.3 

9 

32 

0.662 

21.18 

778 

3.3 

32.3 

"       10 

26 

0.591 

15.36 

551 

3.6 

32.5 

No  bile   exclusion   in   this 

experiment. 

"       11 

38 

0.373 

14.17 

520 

7.3 

32.3 

"       14 

35 

1.071 

37.48 

1,375 

8.5 

32.3 

Large  amount  not  eaten. 

"       15 

34 

0.751 

25.53 

936 

4.1 

32.0 

100  gm.  not  eaten. 

-'•'       16 

14 

0.920 

12.88 

473 

3.4 

32.0 

100    "       "       " 

"       17 

46 

0.630 

28.98 

1,065 

6.4 

32.5 

150    "       "       " 

"       18 

43 

0.822 

35.35 

1,297 

7.5 

32.0 

"       21 

60 

0.759 

45.54 

1,670 

4.2 

32.3 

25      "       "       " 

"       22 

42 

0.453 

19.00 

698 

9.5 

32.0 

Average  

23.84 

875 

5.9 

Diet  0.5  gm.  of  nitrogen  per  kilo. 

Jan.    23 

38 

0.915 

34.77 

1,277 

5.3 

32.0 

571  gm.  of  beef  heart  con- 

taining 100   calories   and 

1.0   gm.   of  nitrogen  per 

kilo. 

;i       24 

45 

0.750 

33.75 

1,238 

8.6 

31.5 

"       25 

62 

0.700 

43.40 

1,593 

5.4 

31.0 

"       28 

35 

0.566 

19.81 

727 

24.2 

30.5 

Diarrhea. 

"       29 

22 

1.400 

30.80 

1,130 

10.3 

30.8 

"       30 

30 

.730 

51.90 

1,905 

7.8 

31.0 

"       31 

46 

.100 

50.60 

1,855 

11.2 

30.8 

Feb.      1 

28 

.394 

29.03 

1,065 

6.8 

30.8 

4 

29 

.131 

32.80 

1,205 

13.6 

31.0 

5 

39 

.015 

39.58 

1,453 

6.0 

30.8 

Noon  meal  omitted. 

6 

12 

1.896 

22.75 

835 

6.3 

31.0 

7 

42 

0.698 

29.31 

1,075 

6.6 

30.5 

8 

29 

0.980 

28.42 

1,042 

11.6 

30.3 

Average  . 

34.37 

1,262 

9.5 

Diet  1.0  gm.  of  nitrogen  per  kilo. 

406 


Metabolism  of  Bile  Acids.     IV 


TABLE  XXXIX- Concluded. 


Amino 

CO 

j 

nitrogen. 

34- 

g|' 

Tt                        1 

Date. 

1 

Inl 
cc.  of 

In  6 

hours. 

ill 

.2.2 

•* 
jf 

a; 

Remarks  . 

> 

bile. 

3 

^ 

1918 

cc. 

mg. 

mg. 

mg. 

mg. 

At. 

Feb.    11 

29 

1.105 

32.05 

1,175 

11.6 

32.0 

209  gm.  of  cracker  meal,  10 

gm.  of  butter,  30  gm.  of 

lard,  65  gm.  of  cane  sugar, 

0.25  gm.  of  nitrogen  and 

100  calories  per  kilo. 

"       12 

23 

0.654 

15.04 

550 

16.0 

31.3 

"       13 

47 

0.403 

18.95 

696 

8.4 

32.0 

"       14 

23 

0.633 

14.56 

534 

36.0 

31.5 

Hb.  124  per  cent. 

•  R.  B.  C.  5,632,000. 

"       15 

48 

0.658 

31.58 

1,160 

10.8 

31.8 

"       18 

45 

0.296 

13.32 

488 

24.3 

30.5 

"       19 

28 

0.331 

9.26 

340 

14.6 

30.5 

"       20 

12 

1.410 

16.92 

620 

13.0 

31.0 

"       21 

17 

0.922 

14.97 

599 

17.2 

31.3 

Average  

18.50 

684 

16.8 

Diet  0.25   gm.   of   nitrogen    per 

kilo. 

very  different  toward  the  same  diet  factor  depending  upon  whether 
a  fasting  period  or  a  carbohydrate  diet  period  had  preceded.  To 
get  a  complete  understanding  of  a  single  diet  factor  it  will  be  neces- 
sary to  observe  any  change  in  the  bile  acid  excretion  curve  which 
may  be  associated  with  the  administration  of  any  such  substance 
after  short  fasting  periods  as  well  as  after  carbohydrate  or  high 
protein  diet  periods. 

Table  XLI  gives  data  to  indicate  that  the  formation  of  bile  acids 
depends  in  part  upon  the  functional  capacity  of  the  liver.  The 
Eck  fistula  liver  is  produced  by  an  anastomosis  between  the  portal 
vein  and  vena  cava  and  a  ligature  on  the  portal  vein  above  this 
anastomosis  which  limits  the  blood  supply  of  the  liver  to  the  he- 
patic artery  and  deviates  all  of  the  portal  blood  directly  into  the  vena 
cava.  The  Eck  fistula  liver  is  known  to  be  smaller  than  normal, 
to  exhibit  a  moderate  degree  of  fatty  degeneration,  to  show  a  marked 
decrease  in  production  of  bile  pigments  (Whipple  and  Hooper,  4) , 
and  to  present  a  distinct  impairment  of  its  normal  capacity  to 


Foster,  Hooper,  and  Whipple 


407 


TABLE  XL. 
Nitrogen  in  Food  and  Bile  Acid  Excretion  after  Fasting  Period. 


Amino 

el   -r 

Dog  No. 

Date. 

«' 

nitrogen. 

|.s  • 

§  £ 

II 

Remarks. 

§ 

In  1 

In  6 

2^2  g 

C.O 

,a 

M 

3 

cc.  of 
bile. 

hours. 

|8- 

|'2 

1 

17-151 

Average    10   days 

fasting  

11.8 

433 

1918 

cc. 

mg. 

mg. 

mg. 

mg. 

Ibs. 

May  4 

37 

0.454 

16.80 

616 

11.0 

32.6 

Diet     330     gm.     of 

cracker    meal,    75 

gm.  of  beef  heart, 

containing  0.5  gm. 

of  nitrogen  and  100 

calories  per  kilo. 

"      6 

19 

0.349 

6.64 

224 

1.6 

36.25 

"      7 

20 

2.4 

35.5 

"      8 

27 

0.367 

9.91 

364 

3.8 

35.8 

Hb.  130  per  cent. 

R.  B.  C.  7,235,000. 

"       Q 

25 

0.350 

8.75 

321 

6.1 

34.7 

"    10 

24 

0.407 

9.77 

359 

6.7 

35.0 

Average  

10.37 

376 

Diet  0.5  gm.  of  nitrogen 

per  kilo. 

15-22 

Average  11    days 

fasting  

11.09 

407 

May  6 

21 

0.587 

12.32 

452 

2.9 

29.2 

Diet     285     gm.     of 

cracker    meal,    60 

gm.  of  beef  heart, 

containing  0.5  gm. 

of  nitrogen  and  100 

calories  per  kilo. 

, 

"      7 

32 

5.6 

29.5 

"      8 

36 

0.353 

12.70 

466 

9.4 

29.2 

Hb.  125  per  cent. 

R.B.C.7,335,.000. 

"      9 

29 

0.365 

10.58 

388 

6.1 

29.0 

"    10 

24 

0.421 

10.10 

371 

4.2 

29.2 

Average 

11.42 

419 

Diet  0.5  gm.  of  nitrogen 

per  kilo. 

408 


Metabolism  of  Bile  Acids.     IV 


excrete  phenoltetrachlorphthalein  (Whipple,  Peightal,  and  Clark, 
5).  We  may  assume  that  the  Eck  fistula  liver  is  functionally 
deficient.  The  observations  in  Table  XLI  harmonize  with  those 

TABLE  XLI. 

Nitrogen  in  Food  and  Bile  Acid  Excretion.    Eck  Fistula. 
Dog  16-15.    Simple  Bile  and  Eck  Fistula. 


Amino 

CO 

fl  0Q 

nitrogen. 

S  3 

Date. 

§ 

"o  a 
"I—  S 

II 

. 

Remarks. 

S 

In  1 

OT3  3 

2 

3 

In  6 

S'S  o 

-      ~ 

1 

cc.  of 
bile. 

hours. 

1" 

1 

1918 

cc. 

mg. 

mg. 

mg. 

mg. 

Ibs. 

Jan.    7 

16 

0.314 

5.03 

183 

4.5 

20.5 

Diet   200    gm.    of    cracker 

meal,     55     gm.     of    beef 

heart,  containing  0.5  gm. 

of      nitrogen      and      100 

calories  per  kilo. 

"       8 

25 

0.258 

6.45 

236 

5.7 

20.0 

Hb.  99  per  cent. 

R.  B.  C.  6,240,000. 

"     10 

12 

0.219 

2.62 

96 

1.9 

19.8 

Not  eating  well. 

"     11 

16 

0.144 

2.30 

84 

5.3 

19.8 

u             «            « 

"     14 

12 

0.155 

1.86 

68 

3.3 

20.0 

"     15 

6 

0.116 

0.69 

25 

20.2 

"     16 

10 

0.141 

1.41 

52 

1.6 

20.7 

"     17 

14 

0.330 

4.62 

169 

3.2 

19.8 

"     18 

21 

0.273 

5.73 

210 

3.7 

20.3 

50  gm.  not  eaten. 

"     21 

20 

0.310 

6.20 

227 

2.1 

20.0 

"     22 

20 

0.212 

4.24 

155 

2.1 

20.0 

Average  

3.74 

137 

3.34 

Diet  0.5  gm.  of  nitrogen  per  kilo. 

Jan.  24 

30 

0.447 

13.41 

492 

3.7 

19.8 

Diet  355  gm.  of  beef  heart, 

containing  1.0  gm.  of  ni- 

trogen and  100  calories  per 

kilo.     1  gm.  of  yeast  twice 

a  day. 

"     25 

17 

0.534 

9.07 

333 

0 

20.8 

outlined  above  and  show  that  an  Eck  fistula  dog  upon  a  standard 
diet  will  excrete  not  over  one-half  the  normal  amount  of  bile  acids. 
Compare  this  dog  (Table  XLI),  weight  20  Ibs.  and  output  on 
standard  diet  of  137  mg.  of  taurocholic  acid  per  6  hour  period,  with 
the  dog  of  Table  XXXV,  weight  40  Ibs.  and  output  on  the  same 


Foster,  Hooper,  and  Whipple  409 

standard  diet  of  550  mg.  of  taurocholic  acid  per  6  hour  period. 
Also  compare  with  dog  of  Table  XXXIX,  weight  33  Ibs.  and  out- 
put on  the  same  standard  diet  of  875  mg.  of  taurocholic  acid  per 
6  hour  period. 

When  the  Eck  fistula  dog  was  changed  to  a  rich  protein  diet  we 
observe  a  considerable  jump  in  the  output  of  bile  acids,  as  in  the 
other  dogs.  It  is  not  possible  to  keep  an  Eck  fistula  dog  on  a  pure 
beef  heart  diet  for  any  length  of  time  without  precipitating  the 
characteristic  Eck  fistula  intoxication  which  usually  results  in 
death. 

DISCUSSION. 

There  is  sufficient  experimental  data  given  above  to  make  the 
point  that  both  endogenous  and  exogenous  factors  are  concerned 
in  the  metabolism  of  bile  acids.  There  is  a  reasonably  constant 
output  of  taurocholic  acid  during  fasting  periods,  and  this  output 
may  be  somewhat  diminished  by  administration  of  pure  carbo- 
hydrate. The  relative  diminution  of  urinary  nitrogen  and  tauro- 
cholic acid  excretion  may  show  a  certain  similarity  under  these 
experimental  conditions.  This  may  indicate  a  certain  relationship 
between  the  metabolism  of  the  body  protein  and  the  production 
of  taurocholic  acid.  There  is  obviously  a  very  important  endog- 
enous factor  in  the  metabolism  of  bile  acids. 

It  is  equally  clear  that  there  is  an  important  relationship  be- 
tween the  output  of  taurocholic  acid  and  the  intake  of  certain  food 
proteins.  On  certain  diets  a  uniform  level  of  bile  acid  excretion 
may  be  observed  for  days  and  a  sudden  shift  to  a  similar  diet  con- 
taining double  the  amount  of  food  nitrogen  may  cause  a  sudden 
doubling  of  bile  acid  excretion.  This  fact  comes  out  clearly  in 
several  experiments  in  this  paper  but  we  wish  to  refer  to  additional 
evidence  submitted  in  Paper  V  of  this  series.  It  is  certain  that 
some  food  proteins  act  very  differently  from  others  as  to  their 
value  in  modifying  the  bile  acid  excretion. 

It  is  to  be  noted  that  the  increased  excretion  of  bile  acids  ap- 
pears very  promptly  when  the  diet  is  changed  to  beef  heart.  It 
may  be  that  the  formation  of  bile  acids  by  the  liver  cell  is  an 
automatic  response  to  the  proper  stimulus,  just  as  these  cells 
respond  to  a  protein  digestion  stimulus  by  urea  formation.  In 
the  case  of  urea  formation  we  know  that  even  in  the  greatest  food 

THE  JOURNAL  OF  BIOLOGICAL  CHEMISTRY,   VOL.  XXXVIII,    NO.  2 


410  Metabolism  of  Bile  Acids.     IV 

shortage  emergency  the  liver  will  form  urea  from  amino-acids, 
even  though  the  body  may  need  all  the  food  amino-acids,  for 
example,  after  long  periods  of  fasting.  In  the  case  of  taurocholic 
acid  we  have  shown  that  long  periods  of  fasting  followed  by  high 
protein  feeding  will  show  little  rise  in  the  taurocholic  acid  output 
contrary  to  what  we  might  expect  if  this  substance  was  purely  a 
waste  product  to  be  eliminated  from  the  split  products  of  protein 
digestion.  The  body  evidently  conserves  the  taurocholic  acid  or 
its  parent  substance  under  certain  conditions,  perhaps  for  use 
elsewhere  in  the  body  in  the  reconstruction  of  its  depleted  body 
protein. 

It  is  generally  accepted  that  bile  acids  are  dependent  upon  the 
ndrrnal  liver  cell  function  for  their  production.  There  has  been 
little  dispute  in  medical  literature  concerning  this  point.  Yet 
there  is  very  little  available  direct  proof  of  this  statement  if  one 
wished  to  argue  that  the  bile  acids  were  formed  elsewhere  in  the 
body  and  eliminated  in  the  bile.  For  example  there  is  convincing 
evidence  that  bile  pigments  may  be  formed  outside  of  the  liver 
and  excreted  in  the  bile  secondarily  (Whipple  and  Hooper). 

We  have  evidence  in  the  Eck  fistula  experiment  given  above  that 
bile  acid  output  will  be  much  subnormal  in  the  Eck  fistula  liver 
which  is  functionally  subnormal.  This  indicates  that  in  a  general 
way  the  bile  acid  output  may  fall  with  impaired  functional  ca- 
pacity of  the  liver  cell.  Other  experiments  in  which  the  liver  is 
injured  by  chloroform  and  other  poisons  give  similar  results  which 
will  be  published  in  the  near  future.  All  this  evidence  gives  some 
direct  and  positive  proof  that  the  bile  acids  are  actually  produced 
by  liver  cell  activity. 

SUMMARY. 

There  is  a  uniform  excretion  of  taurocholic  acid  in  the  bile 
fistula  dog  during  fasting  periods. 

There  is  a  uniform  and  slightly  lower  excretion  of  taurocholic 
acid  in  the  same  dog  during  similar  periods  of  carbohydrate  (sugar) 
feeding.  This  fall  in  taurocholic  acid  excretion  is  much  like  the 
fall  in  urinary  nitrogen  excretion  under  the  same  conditions. 

Th6re  is  an  important  endogenous  factor  in  the  bile  acid 
metabolism  and  this  may  concern  the  body  protein  metabolism. 


Foster,  Hooper,  and  Whipple  411 

The  output  of  bile  acid  in  bile  fistula  bile  may  be  influenced  at 
will  by  suitable  control  of  the  diet.  Meat  protein  seems  to  be  of 
the  greatest  importance  and  a  pure  meat  diet  will  give  the  highest 
output  of  bile  acid  per  6  hour  period. 

There  is  an  important  exogenous  factor  in  bile  acid  metabolism 
which  is  concerned  especially  with  the  food  protein. 

After  a  long  fasting  period  the  bile  fistula  dog  will  not  react  to 
a  high  protein  diet  with  the  usual  high  bile  acid  output.  There 
is  evidently  a  deviation  of  certain  precursors  of  the  bile  acid  to 
serve  other  purposes  in  the  body — perhaps  to  supply  some  im- 
portant substances  relating  to  body  protein  which  have  been 
depleted  by  the  fasting  period. 

A  functionally  deficient  liver  (Eck  fistula)  produces  less  than 
one-half  the  normal  amount  of  bile  acid  during  a  standard  diet 
period.  This  is  direct  evidence  (of  which  there  has  been  little 
available)  that  the  bile  acids  are  formed  essentially  by  liver  cell 
activity. 

BIBLIOGKAPHY. 

1.  Bidder,  F.,  and  Schmidt,  C.,  Die  Verdauungssafte  u.  der  Stoffevechsel, 

Mitau,  1852. 

2.  Kunkel,  A.,  Arch.  ges.  PhysioL,  1877,  xiv,  344. 

3.  Spiro,  P.,  Arch.  PhysioL,  1880,  Suppl.  50. 

4.  Whipple,  G.  H.,  and  Hooper,  C.  W.,  Am.  J.  PhysioL,  1917,  xlii,  544. 

5.  Whipple,  G.  H.,  Peightal,  T.  C.,  and  Clark,  A.  H.,  Johns  Hopkins  Hosp. 

Bull.,  1913,  xxiv,  343. 


THE  WAVERI.Y  P 

BALTIMORE,  U.  6.  A. 


THE  METABOLISM  OF  BILE  ACIDS 

V.    CONTROL  OF  BILE  INGESTION  AND  FOOD 
FACTORS 


BY 

M.  G.  FOSTER,  C.  W.  HOOPER,  AND  G.  H.  WHIFFLE 


(FROM  THE  GEORGE  WILLIAMS  HOOPER  FOUNDATION  FOR  MEDICAL 

RESEARCH,  UNIVERSITY  OP  CALIFORNIA  MEDICAL  SCHOOL, 

SAN  FRANCISCO) 


REPRINTED  FROM 

THE  JOURNAL  OF  BIOLOGICAL  CHEMISTRY 
VOL.  XXXVIII,  No.  2,  JUNE,  1919 


Reprinted  from  THE  JOURNAL  OF  BIOLOGICAL  CHEMISTRY,  Vol.  XXXVIII,  No.  2,  1919 


THE  METABOLISM  OF  BILE  ACIDS. 
V.     CONTROL  OF  BILE  INGESTION  AND  FOOD  FACTORS. 

BY  M.  G.  FOSTER,  C.  W.  HOOPER,  AND  G.  H.  WHIPPLE. 

(From  the  George  Williams  Hooper  Foundation  for  Medical  Research,  Uni- 
versity of  California  Medical  School,  San  Francisco.) 

(Received  for  publication,  April  28,  1919.) 

The  experiments  tabulated  below  serve  two  purposes.  They 
furnish  additional  data  to  make  absolutely  certain  that  the  amount 
of  bile  acid  excreted  can  be  increased  with  the  increase  of  meat 
protein  nitrogen  in  the  diet.  These  experiments  in  addition 
show  that  absolute  exclusion  of  every  drop  of  bile  by  mouth  does 
not  modify  the  excretion  of  bile  acids  under  the  conditions  of  the 
experiment.  It  was  necessary  to  submit  these  control  experi- 
ments because  Stadelmann,  who  has  done  exceptional  experi- 
mental work  in  this  field,  has  stated  that  bile  exclusion  will  cause 
a  fall  of  bile  acid  excretion.  He  assumes  therefore  that  a  muzzle 
at  night  is  necessary  for  accurate  work,  and  that  the  bile  fistula 
dogs  lick  sufficient  bile  from  their  fistulas  to  modify  the  follow- 
ing day's  excretion  of  bile  and  bile  acids. 

Our  dogs  are  set  up  each  morning  after  a  period  of  exercise 
followed  by  a  30  minute  period  of  free  drainage  before  collections 
are  started.  After  the  collection  period  of  6  hours  the  dogs  are  al- 
lowed to  exercise  in  the  yard  before  being  fed  in  their  cages.  They 
are  kept  during  the  night  in  large  cages  of  wire  mesh  beneath 
which  are  pans  for  the  collection  of  excreta.  The  dogs,  of  course, 
during  the  night  drain  bile  from  their  fistulas  into  the  pans,  but 
no  bile  collects  where  the  dogs  have  access  to  it  because  the  wire 
mesh  retains  no  fluids.  The  dogs  usually  lick  their  fistulas  when 
the  collection  is  finished  but  it  seems  to  be  due  in  part  to  the  fact 
that  the  skin  itches  where  it  is  in  contact  with  the  abdominal 
binder.  These  dogs  rarely  lick  bile  from  their  fistulas  in  the 
earry  morning  and  at  this  time  the  flow  is  at  a  minimum.  We 
feel  that  the  experiments  given  make  it  quite  clear  that  under 

413 


414  Metabolism  of  Bile  Acids.     V 

this  laboratory  routine  the  amount  of  bile  which  may  be  obtained 
by  a  given  dog  licking  its  own  fistula  is  not  sufficient  to  modify 
the  excretion  curve  of  bile  acids. 

We  cannot  attempt  to  explain  Stadelmann's  results,  but  two 
factors  may  be  concerned.  It  is  well  known  that  dogs  which 
have  had  bile  fistulas  for  many  months  or  years  do  not  lick  their 
fistulas  as  much  as  do  dogs  who  are  less  accustomed  to  the  bile 
fistula.  One  of  our  dogs  has  been  under  observation  with  a  bile 
fistula  for  3J  years.  Furthermore,  in  our  cages,  as  stated  above, 
bile  could  not  accumulate  during  the  night  where  the  dog  had 
access  to  it  at  any  time,  as  the  bile  flowed  through  the  mesh  cage 
floor.  If  bile  accumulated  on  the  floor  of  a  room  or  cage  it  is 
very  probable  that  a  dog  would  lick  some  of  this  bile  in  the  morn- 
ing and  obtain  a  sufficient  amount  to  make  a  decided  difference 
in  the  output  of  bile  acids. 

It  can  be  deduced  from  the  experiments  given  in  Paper  III 
that  moderate  amounts  of  bile  given  late  in  the  afternoon  will 
not  influence  the  output  of  bile  acids  on  the  following  day.  Bile 
given  in  the  forenoon  will  cause  a  cholagogue  action  lasting 
several  hours,  but  usually  a  rapid  elimination  of  the  excess  of  bile 
acids  within  6  hours — at  least  80  to  90  per  cent  elimination  within 
this  time. 

EXPERIMENTAL. 

Bile  exclusion  in  these  experiments  indicates  that  the  dog  in 
question  was  unable  to  gain  access  at  any  time  to  any  bile  either 
from  its  own  fistula  or  elsewhere.  During  the  routine  6  hour 
collection  all  the  bile  is  collected  in  a  small  rubber  bag.  At  the 
end  of  the  collection  a  muzzle  is  put  securely  on  the  dog,  which 
is  then  permitted  to  run  in  the  yard  for  a  few  minutes  under  care- 
ful observation.  The  dog  is  then  brought  in  and  dressed  with  its 
night  binder  which  is  worn  until  the  next  morning.  The  muzzle 
was  not  worn  during  the  night  except  in  one  instance,  where  the 
dog  tried  to  chew  off  the  straps  of  the  abdominal  binder.  The 
night  binder  was  made  of  light  canvas  and  fitted  to  the  individual 
to  cover  the  thorax  and  abdomen  completely.  Anteriorly  it 
was  held  from  slipping  backward  by  soft  webbing  which  en- 
circles the  neck  and  fore  legs.  The  binder  was  held  about  the 
abdomen  by  soft  webbing  or  straps.  A  large  gauze  pad  was 


Foster,  Hooper,  and  Whipple  415 

placed  over  the  bile  fistula  and  served  to  absorb  all  the  night 
bile.  With  this  routine  we  are  absolutely  certain  that  no  bile 
was  ingested  at  any  time  during  periods  of  "bile  exclusion." 
The  animals  were  comfortable  and  maintained  their  usual  con- 
ditions of  diet  and  activity. 

Tables  XLII,  XLIII,  and  XLIV  are  to  be  considered  as  a 
unit.  These  three  experiments  were  done  at  the  same  time  under 
identical  conditions,  and  the  results  are  remarkably  uniform.  They 
are  to  be  compared  with  Tables  XXXV,  XXXVI,  and  XXXIX 
in  Paper  IV  of  this  series.  In  each  of  the  three  experiments 
tabulated  (XLII  to  XLIV)  the  dog  was  placed  upon  a  diet  of 
cracker  meal  and  beef  heart  containing  0.5  gm.  of  nitrogen  and 
100  calories  per  kilo.  This  diet  with  complete  bile  exclusion 
obtained  for  1  week,  and  the  average  daily  output  per  6  hours 
shows  a  fairly  uniform  figure  of  400  to  500  mg.  of  taurocholic  acid 
per  6  hour  period.  The  dog  which  is  slightly  heavier  shows  a 
slightly  greater  output. 

The  three  dogs  kept  on  this  same  diet  for  the  second  week  were 
not  prevented  from  licking  their  own  fistulas  in  the  cage  during 
the  night.  It  will  be  seen  that  the  bile  acid  output  remains 
about  the  same — it  is  actually  somewhat  less  during  this  second 
week,  340  to  440  mg.  of  taurocholic  acid  per  6  hours. 

During  the  third  week  each  dog  was  given  the  first  day  with  no 
bile  exclusion,  the  second  and  third  day  with  complete  bile  ex- 
clusion, and  the  fourth  day  with  no  bile  exclusion.  Individual: 
fluctuations  appear  but  this  week  in  general  agrees  with  th& 
data  of  the  first  2  weeks. 

The  fourth  week  was  continued  with  strict  bile  exclusion  but 
the  diet  was  changed  to  a  mixture  of  beef  heart  and  a  little  cracker 
meal,  giving  1.0  gm.  of  nitrogen  and  100  calories  per  kilo.  The 
figures  from  this  group  of  experiments  resemble  those  referred 
to  in  Paper  IV.  The  increase  in  beef  heart  stimulates  the  out- 
put of  bile  acids  from  a  level  of  400-500  mg.  to  700-900  mg.  per  6- 
hours. 

Two  points  are  made  by  these  and  other  experiments.  Bile 
exclusion  does  not  modify  the  excretion  of  bile  acids  under  the 
conditions  of  our  experiments.  Certain  food  proteins  in  the  diet 
have  a  marked  influence  on  the  excretion  of  bile  acids  in  bile 
fistula  bile. 


416 


Metabolism  of  Bile  Acids.     V 


TABLE  XLII. 

Known  Diet  With  and  Without  Bile  Exclusion. 
Dog  18-93.     Simple  Bile  Fistula. 


Amino 

nitrogen. 

It 

Date. 

3 

Inl 

In  6 

111 

"S 

Remarks. 

1 

cc.of 
bile. 

hours. 

|8>S 

1 

1918 

CC. 

mg. 

mg. 

mg. 

Ibs. 

Aug.    5 

57  ± 

0.256 

14.59± 

536  ±* 

31.8 

Absolute  bile  exclusion. 

Diet  330  gm.  of  cracker  meal. 

60  gm.  o?  beef  heart  =  0.5 

gm.    of   nitrogen    and    100 

calories  per  kilo. 

"       6 

46 

0.320 

14.70 

540 

31.5 

* 

"       7 

38  ± 

0.254 

9.66± 

355±* 

32.0 

"       8 

56 

0.238 

13.33 

489 

32.0 

"       9 

59 

0.255 

15.05 

552 

32.3 

Average 

13.50 

494 

Complete  bile  exclusion. 

Aug.  12 

54 

0.224 

12.08 

443 

30.8 

No  bile  exclusion. 

"     13 

61 

0.210 

12.81 

470 

31.5 

"     14 

58  ± 

0.242 

14.05^ 

516  ±* 

31.5 

"     15 

61 

0.158 

9.64 

354 

31.3 

"     16 

64 

0.181 

11.58 

425 

31.3 

Average  

12.00 

442 

No  bile  exclusion. 

Aug.  19 

70 

0.266 

18.62 

683 

31.0 

No  bile  exclusion. 

"     20 

71 

0.196 

13.90 

510 

30.5 

Bile  exclusion  12  hours  pre- 

viously. 

"     21 

64 

0.230 

14.72 

540 

30.8 

Bile  exclusion. 

"     23 

77 

0.185 

14.25 

523 

30.5 

No  bile  exclusion. 

Aug.  28 

72 

0.326 

23.48 

862 

30.0 

Absolute       bile       exclusion. 

Diet  467  gm.  of  beef  heart, 

100  gm.  of  cracker  meal  = 

1.0  gm.  of  nitrogen  and  100 

calories  per  kilo. 

«     29 

72 

0.293 

21.08 

774 

29.8 

"     30 

76 

0.335 

25.48 

934 

29.5 

Hb.  126  per  cent. 

R.  B.  C.  6,430,000. 

Average 

23.30 

857 

Complete  bile  exclusion. 

*  2-3  cc.  of  bile  lost. 


Foster,  Hooper,  and  Whipple 


417 


TAB'LE  XLIII. 

Known  Diet  With  and  Without  Bile  Exclusion. 
Dog  18-54-     Bile  Fistula  and  Splenectomy . 


Amino 

nitrogen. 

1-3  • 

Date. 

| 
1 

Inl 
cc.  of 
bile. 

In  6 
hours. 

JiJ 

.C 

i 

Remarks. 

1918 

cc. 

nig. 

mg. 

mg. 

Ibs. 

Aug.    5 

65 

0.170 

11.05 

405 

27.5 

Absolute  bile  exclusion. 

Diet  280  gm.  of  cracker  meal, 

60  gm.  of  beef  heart  =  0.5 

gm.    of    nitrogen    and    100 

calories  per  kilo. 

"       6 

62 

0.18*1 

11.23 

412 

27.8 

"       7 

41 

0.100 

4.10 

150 

28.0 

Binder    chewed    off   night    of 

the  6th. 

"       8 

47 

0.182 

8.56 

314 

27.5 

"       9 

79 

0.212 

16.75 

614 

27.0 

Average             

10.33 

379 

Complete  bile  exclusion. 

Aug.     12 

77  = 

0.224 

17.25=*= 

633 

27.3 

No    bile    exclusion.     5    hour 

collection. 

"       13 

51 

0.140 

7.14 

262 

27.3 

"       14 

43 

0.172 

7.40 

271 

27.0 

"       15 

57 

0.115 

6.56 

241 

27.0 

"       16 

53 

0.126 

6.68 

245 

27.5 

Average  

9.00 

330 

No  bile  exclusion. 

Aug.     19 

77 

0.222 

17.10 

628 

27.5 

No  bile  exclusion. 

20 

76 

0.167 

12.68 

465 

27.3 

Bile    exclusion    12    hrs.    pre- 

viously. 

"       21 

71 

0.187 

13.26 

486 

27.5 

Bile  exclusion. 

"       23 

80 

0.128 

10.24 

376 

27.3 

No  bile  exclusion. 

Aug.    27 

46 

27.0 

Absolute     •  bile        exclusion. 

Diet  417  gm.  of  beef  heart 

and  100  gm.  cracker  meal  = 

1.0  gm.  of  nitrogen  and  100 

calories  per  kilo. 

"       28 

81 

0.288 

23.31 

856 

26.3 

"       29 

75 

0.311 

23.32 

856 

26.5 

"       30 

70 

0.380 

26.60 

976 

26.3 

Hb.  110  per  cent. 

R.  B.  C.  5,515,000. 

Average  .  .  . 

24.40 

896 

Complete  bile  exclusion. 

418 


Metabolism  of  Bile  Acids.     V 


TABLE  XLIV. 

Known  Diet  With  and  Without  Bile  Exclusion. 
Dog  18-137.     Bile  Fistula  and  Splenectomy. 


Date. 

Volume. 

Amino 
nitrogen. 

C  5O 

"o  c 

jM 

tM 

'5 
£ 

Remarks. 

Inl 
cc.  of 
bile. 

In  6 
hours. 

1918 

cc. 

mi. 

mg. 

mg. 

Ibs. 

Aug.    5 

53 

0.313 

16.58 

608 

23.0 

Absolute  bile  exclusion. 

Diet  245  gm.  of  cracker  meal, 

45    gm.    of    beef    heart  =  0.5 

gm.  of  nitrogen  and  100  cal- 

"        6 

56 

0.264 

14.78 

542 

23.0 

ories  pgr  kilo. 

"       7 

38 

0.365 

13.86 

509 

23.3 

"       8 

37 

0.250 

9.25 

340 

23.0 

"       9 

34 

0.198 

6.73 
12.25 

247 

23.0 

Average          -        ... 

450 

Complete  bile  exclusion. 

Aug.  12 

56 

0.196 

10.96 

402 

22.8 

No  bile  exclusion. 

"     13 

46 

0.266 

12.24 

449 

22.3 

"     14 

40 

0.214 

8.56 

314 

22.0 

"     15 

43 

0.129 

5.50 

202 

22.0 

"     16 

44 

0.198 

8.71 

320 

22.0 

Average  .... 

9.20 

338 

No  bile  exclusion. 

Aug.  19 

50 

0.404 

20.20 

742 

22.0 

3  hour  collection. 

"     20 

57 

0.292 

16.65 

611 

22.3 

Bile    exclusion    12    hours    pre- 

viously. 

"     21 

68 

0.188 

12.78 

22.3 

Bile  exclusion. 

"     23 

40 

0.099 

3.96 

145 

22,3 

No  bile  exclusion. 

Aug.  28 

21.8 

Absolute  bile  exclusion.       Diet 

327  gm.  of  beef  heart  and  100 

gm.'  of    cracker    meal  =  1.0 

gm.  of  nitrogen  and  100  cal- 

ories per  kilo. 

"     29 

50 

0.335 

16.75 

611 

22.0 

"     30 

57 

0.376 

21.42 

786 

22.0 

Hb.  120  per  cent. 

R.  B.  C.  5,060,000. 

Average  

19.07 

698 

Complete  bile  exclusion. 

Foster,  Hooper,  and  Whipple 


419 


Tables  XLV  and  XLVI  supply  more  data  on  bile  exclusion 
and  fasting  excretion  of  bile  acids.  These  tables  are  to  be  com- 
pared with  Tables  XXXII  and  XXXIV  in  Paper  IV  and  the 
average  of  these  last  two  tables  is  added  to  Tables  XLV  and 
XLVI  now  under  consideration.  Table  XLV  shows  practically 
the  same  output  of  bile  acids  per  6  hours  whether  bile  is  excluded 
or  not.  This  is  in  harmony  with  all  the  other  experiments  given 
in  this  and  other  papers  as  well  as  many  experiments  which  are 
unpublished. 

TABLE  XLV. 

Bile  Exclusion- — Fasting. 
Dog  17-151.-    Simple  Bile  Fistula. 


Date. 

Volume. 

Amino 
nitrogen. 

O  0 

1C 
f,~  « 

2"23 

3'e  o 

§  C3X 

h 

+2 

II 

W>J3 

'a<o 

•S.2 
M 

1. 
1 

Remarks. 

Inl 
cc.  of 
bile. 

In  6 
hours. 

1918 

cc. 

mg. 

•mg. 

mg. 

mg. 

Ibs. 

May  27 

"      28 

28 
24 

0.693 
0.541 

19.40 
12.98 

712 
476 

26.8 
23,8 

41.2 

Hb.  120  per  cent. 
R.  B.  C.  7,520,000. 

"      29 

19 

0.492 

9.35 

343 

9.3 

39.0 

"      30 

20 

0.558 

11.20 

411 

9.0 

38.0 

"      31 

19 

0.653 

12.41 

455 

13.9 

37.1 

June    1 

23 

0.694 

15.96 

586 

10.0 

36.5 

< 

Average    6  days  .. 

13.55 

497 

Complete  bile  exclusion. 

Average  10  days  .. 

11.42 

419 

No  bile  exclusion.     Table  XXXII. 

Table  XLVI  presents  a  single  contradiction  to  all  the  rest  of  our 
published  data  and  taken  by  itself  would  seem  to  indicate  that 
bile  exclusion  did  actually  diminish  the  bile  acid  output.  Such 
contradictions  are  not  unheard  of  in  experimental  work,  but  we 
believe  this  exception  to  the  general  rule  is  in  reality  explicable. 
This  dog  as  stated  above  (Paper  IV)  is  known  to  have  a  small 
opening  from  his  biliary  tract  into  the  duodenum.  At  times 
he  presents  remarkable  variations  in  the  output  of  bile  acids 
which  stand  in  contrast  to  the  other  dogs  whose  biliary  pass- 
ages are  absolutely  separated  from  the  duodenum.  Because  of 
this  fact  we  attach  little  importance  to  observations  on  this  dog 


420 


Metabolism  of  Bile  Acids.     V 


when  the  bile  is  concentrated  and  viscid,  and  still  less  impor- 
tance to  observations  which  differ  from  those  made  upon  the 
other  fistula  dogs. 

TABLE  XLVI. 

Bile  Exclusion — Fasting. 
Dog  15-22.     Simple  Bile  Fistula. 


Amino 

!« 

nitrogen. 

|.s  • 

a,  ^ 

Date. 

i 

3 

•3 

Inl 

cc.  of 
bile. 

In  6 
hours. 

S73  3 

lls 

l's 

."§  • 

Remarks. 

1918 

cc. 

mg. 

mg. 

mg. 

mg. 

Ibs. 

May  27 

27 

0.905 

24.30* 

892* 

3.7 

34.5 

Hb.  130  per  cent. 

R.  B.  C.  6,540,000. 

"      29 

12 

0.532 

6.38 

234 

14.6 

32.3 

"      30 

21 

0.435 

9.14 

335 

18.3 

31.5 

"     31 

16 

0.380 

6.08 

223 

9.7 

30.9 

June    1 

12 

0.562 

6.74 

247 

14.6 

30.4 

"        2 

20 

0.086 

1.72 

63 

46.0 

29.7 

NH2  determination  repeated 

with  same  result. 

3 

16 

0.510 

8.16 

299 

24.5 

29.4 

Average  6  days  .  .  . 

6.37 

234 

Complete  bile  exclusion. 

Average  9  days  .  .  . 

11.09 

407 

No       bile       exclusion.          Table 

XXXIV. 

*  Not  included  in  average. 


SUMMAKY. 

Complete  exclusion  of  bile  ingestiori  at  all  times  does  not 
modify  the  excretion  of  bile  acids  from  day  to  day.  This  state- 
ment applies  to  the  dogs  used  in  our  experiments  and  to  the  labor- 
atory conditions  under  which  all  this  work  was  done.  It  is  not 
necessary  therefore  to  take  elaborate  precautions  to  prevent  any 
given  dog  from  licking  its  fistula  during  afternoon  and  night 
resting  periods. 

These  experiments  confirm  those  tabulated  in  the  preceding 
paper  to  show  that  an  increase  in  the  meat  proteins  in  the  diet 
will  cause  a  considerable  rise  in  output  of  taurocholic  acid. 


THE  WAVERLY 

BALTIMORE,  U.  8.  A. 


THE  METABOLISM  OF  BILE  ACIDS 
VI.    ORIGIN  OF  TAUROCHOLIC  ACID 


BY 

M.  G.  FOSTER,  C.  W.  HOOPER,  AND  G.  H.  WHIFFLE 


(FKOM  THE  GEORGE  WILLIAMS  HOOPER  FOUNDATION  FOR  MEDICAL 

RESEARCH,  UNIVERSITY  OP  CALIFORNIA  MEDICAL  SCHOOL, 

SAN  FRANCISCO) 


REPRINTED  FROM 

THE  JOURNAL  OF  BIOLOGICAL  CHEMISTRY 
VOL.  XXXVIII,  No.  2,  JUNE,  1919 


Reprinted  from  THE  JOURNAL  OF  BIOLOGICAL  CHEMISTRY,  Vol.  XXXVIII,  No.  2,  1919. 


THE  METABOLISM  OF  BILE  ACIDS. 

VI.     ORIGIN  OF  TAUROCHOLIC  ACID. 
BY  M.  G.  FOSTER,  C.  W.  HOOPER,  AND  G.  H.  WHIPPLE. 

(From  the  George  Williams  Hooper  Foundation  for  Medical  Research, 
University  of  .California  Medical  School,  San  Francisco.) 

(Received  for  publication,  April  28,  1919.) 

Taurocholic  acid  can  be  easily  separated  into  its  two  essential 
constituents,  taurine  and  cholic  acid.  It  has  been  shown  (Paper 
III)  that  in  the  body  the  two  constituent  parts  can  be  easily  and 
rapidly  united  to  form  taurocholic  acid.  This  holds  true  whether 
the  two  substances  are  given  by  mouth  or  whether  cholic  acid  is 
given  by  mouth  and  taurine  intravenously.  It  is  clear  that  cholic 
acid  and  taurine  have  a  strong  physiological  attraction  in  the 
normal  body. 

Taurine  appears  to  be  present  in  the  normal  body  in  a  certain 
excess,  judging  from  cholic  acid  feeding  experiments.  Taurine  is 
more  abundant  in  the  body,  at  least  more  accessible  for  linkage 
with  cholic  acid,  during  full  diet  periods  than  during  fasting  periods 
(Paper  III).  We  have  also  submitted  evidence  to  show  that 
taurine  is  derived  at  least  in  part  from  the  cystine  of  the  food. 
This  is  in  harmony  with  the  observations  of  von  Bergman  (2)  and 
Wohlgemuth  (14),  who  used  somewhat  inaccurate  methods  of 
analysis.  It  is  highly  probable  that  taurine  may  be  derived  from 
other  substances  under  certain  conditions  but  we  have  no  direct 
proof  concerning  this  point.  When  the  metabolism  of  taurine  is 
fully  understood  we  may  be  nearer  a  complete  understanding  of 
the  complex  bile  acid  metabolism. 

Cholic  acid  is  a  substance  which  defies  the  investigator,  who  up 
to  the  present  time  has  learned  little  about  its  source  or  usefulness 
in  the  body.  Cholic  acid  is  of  particular  inter.est  to  us  because  its 
supply  appears  to  be  the  normal  limiting  factor  which  determines 
the  bile  acid  level  of  excretion.  A. sufficient  supply  of  taurine  in 
the  body  seems  to  obtain  under  all  physiological  conditions.  There 
is  sufficient  evidence  to  show  the  important  relationship  that 

421 


422  Metabolism  of  Bile  Acids.     VI 

exists  between  the  basal  nitrogen  metabolism  and  bile  acid 
metabolism.  We  believe  that  knowledge  concerning  the  bile  acid 
metabolism  will  be  of  value  for  a  complete  understanding  of  the 
fundamental  body  protein  metabolism. 

Cholic  acid  is  somewhat  complex  and  its  accepted  formula  is 
given  below.  Because  of  its  structural  formula  many  hypotheses 
have  been  advanced  concerning  its  origin  in  the  body  and  its  pos- 
sible relationship  to  various  drugs.  Some  positive  experiments 
have  been  reported  to  indicate  a  relationship  between  cholic  acid 
and  cholesterol,  but  these  data  will  not  bear  careful  scrutiny.  In 
the  experiments  given  below  we  have  tested  a  number  of  such 
hypotheses  and  without  exception  can  report  only  negative  results. 
It  seemed  to  us  of  some  importance  to  establish  beyond  a  doubt 
that  cholesterol  and  allied  substances  have  no  influence  whatever 
on  the  cholic  acid  metabolism. 

Through  the  work  of  von  Bergman  in  1904,  and  Wohlgemuth  in  the  same 
year,  it  was  clearly  established  that  some  of  the  taurine  of  the  taurocholic 
acid  came  from  cystine.  Both  used  the  sulfur  method  in  their  experi- 
ments. Friedman  (4)  in  1903  had  shown  that  this  change  could  be  effected 
in  vitro,  but  von  Bergman  was  the 'first  to  show  that  when  cystine  is  fed 
to  a  dog  together  with  sodium  cholate  on  a  constant  diet,  there  is  a  marked 
increase  in  the  sulfur  of  the  bile.  There  was  a  distinct  but  less  striking 
increase  when  sodium  cholate  alone  was  given  by  mouth.  There  was  no 
increase  in  bile  sulfur  when  cystine  alone  was  fed.  Wohlgemuth  followed 
the  sulfur  of  the  urine  in  cats  before  and  after  administering  cystine. 
There  was  a  large  amount  of  neutral  sulfur  excreted,  and,  as  there  was  no 
cystine  in  the  urine,  he  concluded  that  all  the  cystine  had  been  changed 
into  taurine.  This  reasoning  may  be  open  to  certain  objections. 

Gibson  (5)  gave  three  injections  of  bromobenzol  to  cats  at  8  to  10  hour 
intervals  to  deflect  the  cystine  from  its  usual  cycle  and  found  a  marked 
decrease  in  the  alcohol-soluble  sulfur  of  the  bile. .  He  does  not  mention 
whether  the  cats  were  kept  upon  a  uniform  diet  nor  does  he  state  their 
clinical  condition  after  administering  the  drug.  The  cats  were  sacrificed  5 
to  8  hours  after  the  last  injection.  We  know  that  this  decrease  in  bile 
sulfur  might  have  been  caused  by  a  diet  low  in  meat  protein  nitrogen,  by 
refusal  of  food,  or  by  the  intoxication  from  the  bromobenzol. 

Lifschiitz  (8)  found  that  cholic  acid  gives  the  same  color  changes  and 
spectrum  as  cholesterol  or  oxycholesterol  when  treated  with  acetic  acid, 
benzoylsuperoxide,  arid  sulfuric  acid.  Flury  (3)  states  that  the  acids 
obtained  by  the  oxidation  of  cholesterol  may  be  placed  in  the  pharma- 
cological group  of  bile  acids  and  saponins  from  their  pharmacological 
behavior.  He  thinks  that  they  may  occur  as  intermediate  steps  in  the 
formation  of  bile  acids  from  cholesterol. 


Foster,  Hooper,  and  Whipple  423 

Excellent  work  has  been  reported  by  Schrotter,  Weitzenbock,  and  Witt 
(11,  12).  They  found  that  cholesterol,  cholic  acid,  oil  of  turpentine,  and 
camphor  all  give  the  same  compound,  rhizocholic  acid,  when  treated  with 
concentrated  sulfuric  acid,  mercury,  and  nitric  acid.  Rhizocholic  acid  is: 


OOH 


HO 


and  was  identified  carefully  in  each  case.     From  this  fact  they  conclude 
that  cholic  acid  and  cholesterol  belong  to  the  terpene  series. 

Pregl  (10)  states  that  cholic  acid  is  closely  related  to  turpentine  and 
camphor  and  is  a  hexahydroxylbenzene.  Moreschi  (9)  in  1913  attempted 
to  show  the  relation  of  cholic  acid  to  cholesterol  by  making  a  trichlor 
derivative  from  each.  The  cholic  acid  formed  a  compound  with  11  atoms 
of  chlorine,  and  cholesterol  with  10  chlorine  atoms, 

Hammarsten  (7)  states  that  in  certain  animals  (shark)  the  bile  acids 
are  replaced  by  scymnol  sulfuric  acid,  made  up  of  a  substance  he  calls 
"scymnol"  which  is  allied  to  cholic  acid  or  cholesterol  paired  with  sulfuric 
acid. 

D'Amato  (1)  found  that  dogs  fed  on  a  lipoid  diet  gradually  increased 
the  excretion  of  both  cholesterol  and  bile  acids  in  the  bile.  The  increase 
noted  in  bile  acids  is  very  small  and  within  the  limits  of  error  of  the  method 
employed  and  surely  within  the  physiological  variations  which  are  observed 
so  frequently  in  routine  collection  periods. 

Goodman  (6)  thought  that  the  broken  down  red  blood  corpuscles  might 
be  available  for  the  formation  of  bile  constituents;  i.e.,  the  cholesterol 
might  be  the  mother  substance  of  cholic  acid.  He  used  a  method  in  which 
he  weighed  the  cholic  acid  excreted.  The  diet  consisted  of  dog  biscuit 
(10  gm.  of  nitrogen  per  day),  varied  with  red  blood  cells,  egg  white,  coagu- 
lated horse  serum,  calves'  brains,  and  cholic  acid.  The  cholic  acid  caused 
a  marked  increase  in  the  cholic  acid  content  of  the  bile,  but  the  bile 
cholesterol  was  unchanged.  This  cholic  acid  increase  might  have  been 
due  to  cholic  acid  excreted  as  such,  and  not  to  taurocholic  acid,  for  his 
method  determines  any  cholic  acid  dissolved  in  the  bile,  whether  united 
with  taurine  or  not.  The  egg  albumin  increased  the  cholic  acid  but  not 
the  cholesterol.  He  used  but  one  dog,  and  that  dog  lived  only  4  weeks. 
For  this  reason  and  because  of  inaccuracies  in  his  methods  we  do  not 
attach  much  importance  to  his  results.  Because  there  is  such  a  small 
amount  of  cholesterol  present  in  the  blood  cells,  Goodman  says  that  in  a 
human  being  there  would  be  necessary  about  60  per  cent  blood  destruction 
per  day,  and  even  more  in  the  dog,  which  of  course  is  unthinkable.  This 
theory  at  best  could  explain  only  a  small  part  of  the  actual  cholic  acid 
production.  Our  experiments  give  no  support  to -this  suggestion. 


424  Metabolism  of  Bile  Acids.     VI 

According  to  Windaus  (13),  the  formula  for  cholesterol  is  as  follows: 

CH3, 

/CxHCH2OH2 CiiHi? 

CH/ 

CH        CH 

H2C        CH         CHCH3 
H2C        CH2        CH 
CHOH  CH2 

Pregl  gives  the  following  formula  for  cholic  acid: — 


CH2    CH2 

COOH— CH/         ^>CH— (CH2)  5— CH 
CHT~CH2 


OH 

EXPERIMENTAL. 

The  experiments  were  conducted  under  conditions  similar  to 
those  described  above.  The  dogs  were  set  up  for  J  hour  for  free 
drainage  of  the  bile  fistula  before  the  collections  were  started. 
Only  a  part  of  the  experimental  data  is  given,  but  it  is  uniform  in 
character  and  the  evidence  all  points  the  same  way. 

Table  L  presents  two  similar  experiments  in  which  cystine  was 
given  intravenously  with  no  positive  influence  on  the  bile  acid 
output,  but  with  a  decided  effect  upon  the  bile  unhydrolyzed 
amino  nitrogen  fraction.  This  may  indicate  an  excretion  in  the 
bile  of  cystine  or  taurine  under  these  conditions.  On  the  follow- 
ing day  a  similar  injection  of  cystine  followed  by  cholic  acid  by 
stomach  resulted  in  a  great  increase  in  taurocholic  acid  excretion 
and  a  return  of  the  unhydrolyzed  amino  nitrogen  to  normal.  This 
gives  evidence  that  under  these  conditions  the  body  can  change 
cystine  rapidly  to  taurine,  which  is  then  available  to  combine  with 
the  cholic  acid.  It  may  be  objected  that  on  a  mixed  diet  the 
feeding  of  cholic  acid  will  increase  the  output  of  taurocholic  acid 
because  plenty  of  taurine  is  available  from  the  food.  Refer  to 
Table  XXIV,  Paper  III,  where  it  is  seen  that  the  rise  in  tauro- 


Foster,  Hooper,  and  Whipple 


425 


cholic  acid  does  actually  reach  a  higher  level  after  administration 
of  4.0  gm.  of  cholic  acid  with  a  full  mixed  diet.  It  would  be  desir- 
able to  repeat  these  observations  during  a  fasting  period. 

Taken  together  with  the  experiments  of  von  Bergman  and 
Wohlgemuth  we  feel  reasonably  secure  in  the  statement  that 
cystine  can  produce  taurine  under  physiological  conditions,  and 

TABLE  L. 

Cystine  Intravenously  Reacts  with  Cholic  Acid. 


Amino  nitro- 

i: 

Dog 

•KJ 

Date. 

• 

a 

gen. 

i;i 

j 

Remarks. 

JNO. 

3 
"g 

In  Ice. 

In  6 

ill 

1 

> 

of  bile. 

hours. 

£* 

1 

1918 

cc. 

mg. 

mg. 

mg. 

Ibs. 

15-22 

Nov.  25 

53 

0.227 

12.04 

442 

33.3 

Mixed  diet. 

"       26 

28 

0.456* 

12.75 

468 

33.0 

0.7  gm.  of  cystine  in- 

travenously. 

,  "       27 

51 

0.746 

38.05 

1,395 

32.5 

0.7  gm.  of  cystine  in- 

travenously   +    2.1 

gm.  of  cholic  acid  by 

stomach. 

17-34 

Nov.  25 

35 

0.198 

6.93 

254 

30.3 

Mixed  diet. 

"       26 

40 

0.356* 

14.24 

522 

30.3 

0.7  gm.  of  cystine  in- 

travenously.    Vom- 

ited. 

"       27 

76 

0.453 

34.45 

1,264 

30.3 

0.7  gm.  of  cystine  in- 

travenously   +    2.1 

gm.  of  cholic  acid  by 

stomach.  Vomited  a 

little  of  cholic  acid 

solution. 

*  The  unhydrolyzed  bile  gave  a  high  unhydrolyzed  NH2  showing  the 
presence  of  an  excess  of  cystine  or  taurine  uncombined  with  cholic  acid. 
The  following  day  when  cholic  acid  was  given  this  unhydrolyzed  NH2  had 
returned  to  normal  which  would  indicate  that  all  the  cystine  Had  been 
synthesized  into  taurocholic  acid. 

this  taurine  is  available  to  combine  with  an  excess  of  cholic  acid. 
This  holds  good  apparently  whether  the  cystine  is  fed  by  mouth 
(von  Bergman)  or  given  intravenously  (Table  L). 

In  comparing  Table  LI  with  Tables  XXXI  and  XXXIII  of 
Paper  IV,  one  can  see  that  these  variations  are  within  the  normal 
fluctuations  of  these  same  dogs  on  a  sugar  diet. 


THE   JOURNAL   OP    BIOLOGICAL  CHEMISTRY,    VOL     XXXVIII,  NO     2 


426 


Metabolism  of  Bile  Acids.     VI 


Tables  LI  and  LII  are  to  be  considered  together  and  they  furnish 
strong  evidence  that  cholesterol  alone  or  fed  with  taurine  exerts  no 
influence  upon  the  excretion  of  taurocholic  acid.  This  holds  for 
periods  of  fasting  as  well  as  for  periods  of  mixed  diet.  The 
amounts  of  cholesterol  administered  (3  to  4  gm.)  are  sufficient  to 
convince  any  investigator  that  this  substance  does  not  play  a  part 
in  bile  acid  metabolism.  There  is  no  immediate  reaction  nor  any 

TABLE  LI. 
Cholesterol  Feeding — Sugar  Diet — Bile  Acids  Unchanged. 


Amino 

3 

CO 

A 

nitrogen. 

1 

C 

a 

Dog 
No 

Date. 

-o 

£ 

;-   S 
O   3 

•3° 

>>  09 

Remarks. 

G 

3 

0_QJ 

3 

Ss 

C3  3 

"S 

O 

5" 

G 

hH 

;H 

^ 

P 

O) 

1918 

CC. 

mg. 

m0. 

m<7. 

mg. 

gm. 

»s. 

17-151 

Average  11  days. 

8.05 

295 

3.45 

Diet  of  75  gm.  of  cane 

sugar,    100    gm.    of 

glucose. 

Apr.  6 

15 

0.402 

6.03 

220 

2.52 

35.0 

«     7 

17 

0.556 

9.45 

347 

18.6 

2.13 

35.1 

4  gm.  of  cholesterol.* 

"     8 

17 

0.416 

7.08 

260 

14.2 

2.57 

34.4 

18-23 

Average  11  days. 

8.66 

318 

3.23 

Diet  of  75  gm.  of  cane 

sugar,  75  gm.  of  .glu- 

cose. 

Apr.  6 

37 

0.171 

6.32 

232 

14.2 

3.30 

25.8 

"     7 

31 

0.269 

9.34 

343 

13.0 

2.58 

25.7 

4  gm.  of  cholesterol.* 

"     8 

37 

0.125 

4.62 

169 

18.7 

2.18 

25.7 

*  Given  in  gelatin  capsule  at  beginning  of  6  hour  collection. 

delayed  effect  to  be  observed.  In  Table  LII  the  taurine  was  given 
to  insure  an  excess  of  this  substance  in  the  body  to  combine  with 
any  amount  of  cholic  acid  available.  Table  LII  illustrates  the 
inhibition  of  bile  flow  due  to  sugar  by  mouth  (Dog  15-22). 
Vomiting  on  certain  occasions  may  be  associated  with  a  decreased 
flow  of  bile  in  these  fistula  dogs.  The  amount  of  ether  used  to 
dissolve  the  cholesterol  (8  to  10  cc.)  will  have  no  influence  upon 
the  bile  excretion. 


Foster,  Hooper,  and  Whipple 


•427 


Tables  LIII  and  LIV  are  similar  experiments  which  show  that 
red  blood  cells  by  mouth  have  no  effect  upon  the  excretion  of  bile 
acids.  The  control  periods  of  5  days  and  the  red  blood  cell  feed- 
ing periods  give  figures  which  are  practically  identical.  The 

TABLE  LII. 

Taurine  Plus  Cholesterol  Feeding — Bile  Acids  Unchanged. 


Amino 

o 

nitrogen. 

I.S 

, 

Dog 

Date. 

0) 

if"  2 

j 

Remarks. 

S 

j3 

In  1 

cc.  of 
bile. 

In  6 
hours 

111 

1 

1918 

CC, 

mg. 

mg. 

mg. 

Ibs. 

Mixed  diet. 

18-93 

Sept.  11 

76 

0.210 

15.95 

586 

31.5 

"     12 

58 

0.320 

18.55 

681 

32.5 

1  gm.  of  taurine,*  3  gm.  of 

cholesterol.   Some  diarrhea. 

r 

Intoxicated  by  ether. 

"     13 

76 

0.225 

17.10 

628 

34.0 

Hb.  126  per  cent. 

R.  B.  C.  6,430,000. 

15-22 

6.40 

235 

Average  10  days  fasting. 

June    6 

3.6 

0.642 

2.31 

84 

28.2 

100  gm.  of  cane  sugar  and  25 

gm.  of  glucose  after  10  days 

fasting  with  bile  exclusion. 

Vomiting. 

"      7 

10 

0.616 

6.16 

226 

27.4 

2  gm.  of  taurine,*  3  gm.  of 

cholesterol. 

Mixed  diet. 

18-54 

Sept.    3 

12 

0.170 

7.14 

262 

27.0 

Hb.  110  per  cent. 

R.  B.  C.  5,515,000. 

"       4 

46 

0.141 

6.49 

238 

27.4 

1  gm.  of  taurine*  and  3  gm.  of 

cholesterol.     Intoxicated  by 

ether. 

"       5 

58 

0.113 

6.56 

241 

28.0 

*  Taurine  dissolved  in  water.     Cholesterol  dissolved  in  ether  and  given 
by  stomach  tube  at  beginning  of  6  hour  collection. 

mixed  diet  gives  considerable  daily  fluctuation,  but  the  averages 
are  nearly  uniform.  The  addition  of  10  gm.  of  dried  red  blood 
cells  does  not  influence  the  curve  of  bile  acid  excretion.  This 
amount  of  red  cells  contains  an  appreciable  amount  of  cholesterol, 
but  of  course  not  comparable  to  the  large  amounts  used  in  Tables 


428* 


Metabolism  of  Bile  Acids.     VI 


LI  and  LII.     We  have  other  experiments  with  brain  feeding  which 
show  the  same  negative  influence  on  bile  acid  excretion. 

Table  LV  shows  the  negative  reaction  following  the  intravenous 
injection  of  laked  red  blood  cells.  This  again  illustrates  how 
easily  we  may  dissociate  the  excretion  curves  of  bile  pigments  and 

TABLE  LIII. 

Red  Blood  Cell  Feeding' — Bile  Acids  Unchanged. 
Dog  18-23.     Simple  Bile  Fistula. 


Amino 

. 

nitrogen. 

I.S  • 

Date. 

3 

Inl 

In  6 

22  5 

bt 

Remarks. 

I 

cc.  of 
bile. 

hours. 

§  i-S 

1 

1918 

CC. 

mg. 

mg. 

mg. 

Ibs. 

July  15 

56 

0.272 

15.23 

559 

31.8 

Mixed    diet.    Absolute    bile    exclu- 

sion. 

"     16 

63 

0.254 

16.00 

587 

33.3 

Hb.  115  per  cent.-    R.  B.  C.  6,200,000. 

"     17 

67 

0.297 

19.90 

731 

32.5 

"     18 

71 

0.410 

29.10 

1,068 

32.0 

"     19 

62 

0.324 

20.18 

741 

32.3 

Average  

20.00 

734 

July  22 

53 

0.254 

13.47 

495 

31.0 

Mixed  diet  +  10  gm.  of  R.  B.  C.* 

"     23 

60 

0.262 

15.72 

576 

32.3 

it          tt    +  10    «      «          « 

"     24 

57 

0.302 

17.20 

628 

32.5 

«          tf     i    -if\    K      «          u 

"     25 

64 

0.271 

17.35 

637 

32.8 

<(                      «         _J_    1Q         «              «                      « 

"     26 

62 

0.326 

20.20 

742 

«                      ..             1        1Q          .,              «                      « 

Averag 

B                  ... 

16.80 

617 

*  Prepared  by  washing  red  blood  cells  in  normal  saline  3  times.  The 
residual  cells  were  dried  by  warm  air  and  ground  to  a  powder,  10  gm.  of 
red  cells  made  into  an  indefinite  solution  emulsion  mixture,  flavored  with 
a  little  sugar  and  salt,  made  up  to  400  cc.  with  water,  and  given  by 
stomach  tube. 

bile  acids.  The  intravenous  injection  of  large  amounts  of  laked 
red  blood  cells  will  cause  a  prompt  and  large  increase  in  the  output 
of  bile  pigments  but  110  increase  in  bile  acid  excretion.  There  is 
no  immediate  and  no  delayed  bile  acid  reaction  which  we  have  been 
able  to  observe.  Some  investigators  have  recorded  a  drop  in  bile 
acid  excretion  following  intravenous  injections  of  hemoglobin  but 
our  experiments  are  clear  cut  and  negative  in  this  respect.  One 


Foster,  Hooper,  and  W  hippie 


429 


observes  only  the  normal  physiological  fluctuations.  A  possible 
explanation  of  the  observed  depression  of  bile  acid  excretion  is.  the 
febrile  reaction  which  sometimes  is  observed  following  hemoglobin 
injections.  No  such  reaction  was  observed  in  these  experiments. 
It  is  well  to  note  that  the  whole  laked  cells  were  given — that  is, 
stroma  and  hemoglobin.  After  the  blood  had  been  laked  by  dis- 

TABLE  LIV. 

Red  Blood  Cell  Feeding- — Bile  Acids  Unchanged. 
Dog  18-54.    Bile  Fistula  and  Splenectomy. 


Amino 

nitrogen. 

*O  fl 
fe+  .^- 

g 

3 

Inl 

In  6 

83s 

43 
hO 

! 

cc.of 
bile. 

hours. 

£** 

3 

' 

1918 

CC. 

mg. 

mg. 

mg. 

Ibs. 

July  22 

37 

0.268 

9.91 

363 

28.0 

Mixed  diet.     Bile  exclusion. 

"     23 

41 

0.213 

8.71 

319 

28.3 

"     24 

18 

0.315 

5.67 

208 

27.8 

"     25 

33 

0.299 

9.87 

362 

27.5 

"     26 

45 

0.282 

12.68 

466 

Average 

9  40 

345 

July  30 

65 

0.125 

8.13 

299 

28.0 

Hb.  100  per  cent. 

R.  B.  C.  5,075,000. 

Mixed  diet  +  10  gm.  of  R.  B.  C.* 

"     31 

48 

0.237 

11.36 

417 

28.3 

"          "    +  10     "    "            " 

Aug.   1 

50 

0.284 

14.20 

522 

27.8 

((               «        i     -ir\        «      ((    .             « 

"       2 

37 

0.145 

5.36 

196 

27.5 

«    +10     «    « 

Tube  out.    About  10  cc.  lost. 

Average  

9.7 

356 

*  Red  blood  cells  prepared  as  in  Table  LIII. 

tilled  water  it  was  shaken  thoroughly  to  insure  a  complete  injec- 
tion of  stroma  and  cell  fragments.  It  has  been  suggested  repeat- 
edly that  the  red  cell  stroma  and  perhaps  the  contained  cholesterol 
were  waste  products  which  normally  came  to  the  liver  to  be  trans- 
formed into  bile  acids  and  eliminated  in  the  bile.  This  is  an 
attractive  hypothesis  which  is  delightfully  simple,  but  like  many 
others  it  has  no  basis  of  experimental  fact  and  should  be  put  aside 
even  if  with  regret. 


430 


Metabolism  of  Bile  Acids.     VI 


Since  Schrotter,  Weitzenbock,  and  Witt  were  able  to  make 
rhizocholic  acid  from  cholic  acid,  cholesterol,  turpentine,  or 
camphor,  we  thought  it  might  be  possible  to  produce  cholic 
acid  in  the  animal  body  by  feeding  either  turpentine  or  camphor. 
Taurine  was  given  on  the  last  day  in  each  experiment  (Table  LVI) 
so  that  any  cholic  acid  which  might  be  formed  in  the  organism 
would  unite  with  the  taurine  and  be  excreted  in  the  bile  as  tauro- 

TABLE  LV. 

Laked  Red  Cells  Intravenously' — Bile  Acids  Unchanged. 


Ami  no 

nitrogen. 

.2  o 

Dog 
No. 

Date. 

a 

la  I 

Remarks. 

In  1 

3 

cc.  of 
bile. 

In  6 
hours. 

JSJ 

^ 

1918 

cc. 

mg. 

mg. 

mg. 

Ibs. 

18-23 

July  29 

66 

0.262 

17.28 

637 

30.5 

Mixed  diet. 

"     30 

72 

0.223 

16.05 

590 

32.3 

Laked  blood.* 

"     31 

58 

0.349 

20.25 

744 

32.3 

Hb.  120  per  cent. 

R.B.C,  6,375,000. 

Aug.     1 

63 

0.287 

18.18 

678 

31.8 

Laked  blood.* 

"       2 

51 

0.413 

21.03 

772 

32.3 

18-93 

July  29 

68 

0.207 

14.07 

517 

30.3 

Mixed  diet. 

"     30 

66 

0.241 

15.90 

584 

31.8 

Laked  blood.* 

"     31 

53 

0.280 

14.85 

545 

32.3 

Hb.  135  per  cent. 

R.  B.  C.  6,380,000. 

Aug.    1 

70 

0.229 

16.02 

588 

32.3 

Laked  blood.* 

"       2 

64 

0.315 

20.15 

740 

33.3 

*  100  cc.  of  sterile  defibrinated  normal  blood,  centrifuged,  red  cells 
•washed  and  laked  with  water,  made  up  to  100  cc.,  and  kept  on  ice  over 
night;  rendered  isotonic,  warmed,  shaken,  and  given  intravenously  by 
hypodermic  needle  2  hours  after  start  of  collection. 

cholic  acid.  This  was  probably  an  unnecessary  precaution  when 
we  recall  the  great  output  of  taurocholic  acid  which  follows  a  cholic 
acid  feeding  during  periods  of  mixed  diet.  This  indicates  an 
abundant  source  of  taurine  in  the  body  during  periods  of  liberal 
feeding,  more  than  enough  to  combine  with  any  expected  excess 
of  cholic  acid. 

Careful  scrutiny  of  Table  LVI  reveals  no  fluctuations  other  than 
those  observed  in  control  periods,  whether  terpene  hydrate  alone 
or  combined  with  taurine  was  given  by  stomach.  There  is  no 


Foster,  Hooper,  and  Whipple 


431 


•  TABLE  LVI. 
Terpene  Hydrate  With  and  Without  Taurine  by  Mouth. 


Amino 

nitrogen. 

^5° 

Dog 

No. 

Date. 

flj 

1 

l-sf 

'So 

Remarks. 

Inl 

In  6 

"o 

cc.  oi 
bile. 

hours 

Is-2 

'* 

1918 

cc. 

mg. 

mg. 

mg. 

Z6s. 

Mixed  diet. 

15-22 

Sept..  3 

22 

1.150 

25.30 

929 

33.3 

Hb.  150  per  cent.  R.  B.  C. 

7,455,000. 

"       4 

47 

0.214 

10.05 

369 

34.5 

1  gm.  of  taurine,  1  gm.  of  ter- 

pene hydrate.* 

«                K 

45 

0.381 

17.15 

630 

33.5 

1  gm.  of  taurine,  1  gm.  of  ter- 

* 

pene  hydrate.* 

"       6 

38 

0.388 

14.73 

541 

32.8 

Mixed  diet.           •   ' 

18-23 

Aug.   27 

57 

0.261 

14.86 

545 

30.8 

"     28 

57 

0.343 

19.55 

718 

31.8 

0.25  gm.  of  terpene  hydrate.* 

"     29 

70 

0.309 

21.63 

794 

33.3 

0.5  gm.  of  terpene  hydrate.* 

0.5  gm.  of  taurine. 

•"     30 

53 

0.434 

23.00 

844 

33.3 

Mixed  diet. 

18-54 

Sept.  10 

61 

0.112 

6.84 

751 

28.8 

"     11 

82 

0.238 

19.50 

716 

29.0 

"     12 

81 

0.186 

15.05 

552 

28.5 

1    gm.   of   terpene    hydrate,* 

1  gm.  of  taurine  by  stomach 

tube.     Diarrhea. 

"     13 

47 

0.155 

7.29 

267 

27.8 

Mixed  diet. 

17-181 

Sept.  10 

49 

0.292 

14.30 

525 

25.0 

"     11 

42 

0.280 

11.75 

431 

25.3 

"     12 

67 

0.267 

17.85 

655 

26.0 

1  gm.  of  taurine,  1  gm.  of  ter- 

pene hydrate.*     Diarrhea. 

"     13 

42 

0.324 

13.60 

499 

25.0 

. 

Mixed  diet. 

18-93 

Sept.    3 

64 

0.285 

18.25 

670 

30.5 

Hb.  126  per  cent.  R.  B.  C. 

6,430,000. 

"       4 

44 

0.073 

3.94 

144 

33.5 

1  gm.  of  taurine  and  1  gm.  of 

terpene  hydrate.*  Diuresis. 

"       5 

54 

0.183 

9.89 

363 

32.5 

1  gm.  of  taurine,  1  gm.  of  ter- 

pene hydrate.* 

"       6 

64 

0.291 

8.61 

684 

*  Terpene  hydrate  dissolved  in  a  little  dilute  alcohol ;  taurine  dissolved 
in  water,  given  by  stomach  tube  at  beginning  of  6  hour  collection. 


432 


Metabolism  of  Bile  Acids.     VI 


cholagogue  action  and  no  change  in  the  bile  concentration  and 
total  amounts  of  bile  acids. 

Table  LVII  shows  the  results  of  administration  of  camphor  by 
mouth.  Repeated  doses  of  spirits  of  camphor  have  no  effect  upon 
the  bile  acid  excretion.  Similar  observations  have  been  made  on 

TABLE  LVII. 

Camphor  Feeding  With  and  Without  Taurine  by  Mouth. 
Dog  15-22.     Simple  Bile  Fistula. 


Date. 

Volume. 

Amino 
nitrogen. 

Taurocholic 
acid  in  6 
hours. 

js 

M 

i 

Remarks. 

Inl 
cc.of 
bile. 

In  6 
hours. 

1918 

cc. 

mg. 

mg. 

mg. 

Ibs. 

July  29 

48 

0.290 

13.92 

511 

33.5 

Mixed  diet. 

"     30 

29 

0.155 

4.49 

165 

34.3 

0.2  gm.  of  camphor*  by  stomach  tube. 

Hb.  135  per  cent.     R.  B.  C.  6,430,000. 

"     31 

35 

0.292 

10.22 

375 

34.3 

0.4    gm.    of    camphor    by    stomach 

tube. 

Aug.    1 

38 

0.430 

16.34 

600 

33.5 

0.6    gm.    of    camphor    by    stomach 

tube. 

Considerable  salivation. 

"       2 

14 

1.030 

14.41 

529 

33.5 

"       5 

47 

33.8 

0.4    gm.    of    camphor    by    stomach 

tube. 

"       6 

35.0 

0.4    gm.    of    camphor    by    stomach 

tube. 

"       7 

53 

0.269 

14.25 

523 

34.3 

0.4    gm.    of    camphor    by    stomach 

tube. 

"       8 

63 

0.222 

14.00 

514 

33.3 

0.4  gm.  of  camphorand.l  gm.taurine 

by  stomach  tube. 

"       9 

40 

0.439 

17.56 

644 

34.0 

0.4    gm.    of    camphor    by    stomach 

tube. 

*  Camphor  given  as  a  10  per  cent  solution — spirits  of  camphor.  Diu- 
resis was  uniformly  noted. 

other  dogs.     Obviously  there  is  no  immediate  and  no  delayed 
reaction  which  can  be  attributed  to  the  action  of  this  drug. 

It  is  evident  that  neither  camphor  nor  terpene  hydrate  have  any 
effect  on  the  excretion  of  taurocholic  acid.  Cholesterol,  turpen- 
tine, cholic  acid,  and  camphor  may  all  belong  to  the  same  group 
chemically,  but  physiologically  they  certainly  are  not  closely 
related,  at  least  as  regards  bile  acid  metabolism. 


Foster,  Hooper,  and  Whipple  433 

SUMMARY. 

Taurine  as  found  in  the  body  is  derived  in  all  probability  from 
the  cystine  of  the  food  or  body  protein.  Taurine  appears  to  be 
present  in  excess  of  the  amount  needed  to  combine  with  the  cholic 
acid  of  normal  metabolism. 

Cholic  acid  is  the  limiting  factor  which  determines  the  level  of 
bile  acid  excretion  in  the  bile.  The  origin  and  fate  of  cholic  acid 
have  not  been  satisfactorily  determined. 

Cholesterol  fed  alone  or  combined  with  taurine  causes  no  change 
in  the  excretion  of  bile  acids.  This  gives  no  evidence  of  any  physi- 
ological relationship  between  cholesterol  and  cholic  acid. 

Red  blood  cells  fed  by  mouth  or  hemolyzed  and  injected  intra- 
venously have  no  influence  upon  the  level  of  bile  acid  excretion. 

Terpene  hydrate  and  camphor  fed  alone  or  combined  with 
taurine  do  not  influence  the  curve  of  bile  acid  excretion. 

BIBLIOGRAPHY. 

1.  d'Amato,  L.,  Biochem.  Z.,  1915,  Ixix,  217. 

2.  von  Bergman,  G.,  Beitr.  chem.  Physiol.  u.  Path.,  1904,  iv,  192. 

3.  Flury,  F.,  Arch.  Exp.  Path.  u.  Pharm.,  1911,  Ixvi,  221. 

4.  Friedman,  E.,  Beitr.  chem.  Physiol.  u.  Path.,  1903,  iii,  25. 

5.  Gibson,  R.  B.,  J.  Biol.  Chem.,  1909,  vi,  p.  xvi. 

6.  Goodman,  E.  H.,  Beitr.  chem.  Physiol.  u.    Path.,  1907,  ix,  91. 

7.  Hammarsten,  O.,  Z.  physiol.  Chem.,  1898,  xxiv,  322. 

8.  Lifschiitz,  J.,  Ber.  chem.  Ges.,  1914,  xlvii,  1459. 

9.  Moreschi,  Redn.  soc.  chim.  ital.,  1913,  v,  242. 

10.  Pregl,  F.,  Z.  physiol.  Chem.,  1910,  Ixv,  157. 

11.  Schrotter,  H.,  Weitzenbock,  R.,  and  Witt,  R.,  Monatshr.  Chem.,  1908, 

xxix,  245. 

12.  Schrotter,  H.,  and  Weitzenbock,  R.,  Monatschr.  Chem.,  1908,  xxix,  395. 

13.  Windaus,  A.,  Uber  Cholesterin;  Habilitationsschrift,  Freiburg  i.     Br. 

Speyer  u.  Karner,  1903-Sep:  v.  Vf. 
Abstracted  in  Chem.  Zentr.,  1903,  i,  814. 

14.  Wohlgemuth,  J.,  Z.  physiol.  Chem.,  1903-04,  xl,  81. 


THE  WAVERLY  PRESS 
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